
==== Front
Stem Cell Reports
Stem Cell Reports
Stem Cell Reports
2213-6711
Elsevier

S2213-6711(24)00112-7
10.1016/j.stemcr.2024.04.006
Article
Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis
Huang Cheng 1235
Jiang Haiping 1235
Dong Jingxi 1245
Jiang Liyuan 1
Li Jie 123
Xu Jing 123
Cui Tongtong 124
Wang Leyun 14
Li Xin 124
Feng Guihai 124
Zhang Ying 124
Li Tianda litianda@ioz.ac.cn
124∗
Li Wei liwei@ioz.ac.cn
12346∗∗
Zhou Qi zhouqi@ioz.ac.cn
1234∗∗∗
1 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
2 Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China
3 University of Chinese Academy of Sciences, Beijing 100049, China
4 Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
∗ Corresponding author litianda@ioz.ac.cn
∗∗ Corresponding author liwei@ioz.ac.cn
∗∗∗ Corresponding author zhouqi@ioz.ac.cn
5 These authors contributed equally

6 Lead contact

09 5 2024
11 6 2024
09 5 2024
19 6 877889
18 3 2024
14 4 2024
15 4 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived in vitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT.

Highlights

• Mouse hepatocytes were isolated successfully from mouse-rat chimeric livers

• Interspecies chimera-derived hepatocytes exhibited mature hepatic function

• Chronic liver fibrosis was alleviated after chimeric hepatocyte transplantation

In this article, Zhou and colleagues show that mouse hepatocytes were isolated successfully from chimeric livers in mouse-rat interspecies chimeras. Chimeric hepatocytes exhibited mature hepatic function and were similar to endogenous hepatocytes. Mouse chronic liver fibrosis was alleviated after interspecies chimera-derived hepatocyte transplantation. Their results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes for transplantation.

Keywords

Interspecies chimeras
Blastocyst complementation
Hepatocytes
Hepatic functions
Chronic liver fibrosis
Published: May 9, 2024
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pmcIntroduction

The liver disease represents an important public health burden worldwide. Liver transplantation (LT) is the only effective therapy when liver disease progresses to liver failure; however, the shortage of donors is a significant issue (Xie et al., 2021). In theory, hepatocyte transplantation (HT) is a potential alternative to LT because transplanted hepatocytes can compensate for impaired hepatic function (Iansante et al., 2018). However, hepatocytes for transplantations require high quality and sufficient quantity along with ethical issues; therefore, reliable alternative sources are needed (Ruoß et al., 2020; Zeilinger et al., 2016). Potential alternative hepatocytes can be obtained via differentiation of pluripotent stem cells (PSCs) (Baxter et al., 2015; Yu et al., 2012), transdifferentiation (Du et al., 2014; Huang et al., 2014; Kogiso et al., 2013), the expansion of primary human hepatocytes (Zhang et al., 2018), and humanized livers in animal models (Azuma et al., 2007; Bissig et al., 2007; Ren et al., 2022). However, these approaches for the generation of hepatocytes still face challenges such as low hepatocyte maturity or the essentiality of primary hepatocytes as starting cells.

Interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC), which refers to the injection of donor PSCs of one species into blastocysts of another species, may be a promising donor hepatocyte source (Rashid et al., 2014; Wu et al., 2016). The generation of hepatocytes by IBC is precisely related to the optimal induction of hepatocyte differentiation in vivo as IBC may provide preferable hepatocytes with all conditions and factors necessary for cell development (Xie et al., 2021). In addition, IBC is not limited by the supply of primary hepatocytes and requires only a few PSCs for hepatocyte generation.

The generation of interspecies chimeras via IBC is a potentially effective approach for generating cells, tissues, and organs. To date, IBC-derived organs have been successfully created in the pancreas (Kobayashi et al., 2010; Yamaguchi et al., 2017), thymus (Isotani et al., 2011), kidney (Goto et al., 2019), heart (Coppiello et al., 2023), and other organs (Wen et al., 2022; Wu et al., 2017) between mice and rats, suggesting that IBC may have potential application in regenerative therapy. Previous studies have shown that chimeric hepatocytes are observed in interspecies chimeras, with their identification predominantly relying on polymerase chain reaction (PCR), immunostaining techniques targeting albumin (ALB), or the drug-metabolizing enzyme, CYP2C6 (Isotani et al., 2011; Kobayashi et al., 2010; Li et al., 2018; Nishimura et al., 2021; Okumura et al., 2019; Wu et al., 2017). However, this evidence is not sufficient to reflect complete hepatic function, normal transcriptome, and recovery capability for related liver diseases. These characteristics of chimeric hepatocytes are critical for future clinical application in the treatment of liver diseases.

Following generation of mouse-rat interspecies chimeras using IBC and isolation of mouse embryonic stem cell (ESC)-derived hepatocytes from livers, we aimed to perform hepatic function identification such as lipid accumulation, glycogen storage, urea synthesis, and transcriptome analysis of interspecies chimera-derived hepatocytes and also investigate their potential application in the treatment of chronic liver fibrosis. Our goal was to investigate whether chimeric hepatocytes could be a reliable source of hepatocytes and be applied as a therapeutic method for HT.

Results

Mouse ESCs robustly contributed to mouse-rat interspecies chimeras with a wide range of tissues and organs

To generate mouse-rat interspecies chimeras using IBC, we established two different mouse ESC lines from 129 and C57 strains of mice carrying green fluorescent protein (GFP) for cell tracking (Figures 1A, 1B, and S1A). Next, we injected GFP-mouse ESCs into rat blastocysts and observed fluorescent markers in chimeric blastocysts (Figures 1C and S1B). We then transplanted chimeric blastocysts into the uterus of pseudo-pregnant rats, and the embryos at embryonic day (E) 13.5 showed a high percentage of chimeric contribution (Figure S1C). The proportion of chimeric fetuses by the contribution of both mouse ESC lines tested was approximately 14%–18% (Table 1). Both 129 and C57 mouse ESC lines gave rise to adult chimeras with partial black or brown coat colors (Figure 1D).Figure 1 Mouse ESCs robustly contributed to mouse-rat interspecies chimeras with a wide range of tissues and organs

(A) Schematic of generated mouse-rat chimeras obtaining stable chimeric organs by injecting GFP mouse ESCs into rat blastocysts.

(B) GFP-positive mouse ESCs derived from 129 mouse strain. Scale bar, 100 μm.

(C) Blastocyst-stage Sprague-Dawley (SD) strain rat embryos injected with GFP-positive 129 strain mouse ESCs. Scale bar, 50 μm.

(D) Interspecies chimeric adult at 8 weeks (left), with wild-type SD rat at 8 weeks (right).

(E) Fluorescence images of organs derived from chimeras. Mouse ESCs with GFP expression were found in the heart, liver, kidneys, lung, brain, and spleen. Scale bar, 2 mm.

(F) Determination of the cell contribution of mouse ESCs by PCR analysis of mouse Gapdh (mGapdh) and rat Gapdh (rGapdh). Mouse and rat genomes are used as controls. (n = 5 mouse-rat chimeras; 6 weeks old).

(G) Levels of chimerism of mouse ESCs in different solid organs of 6-week-old mouse-rat chimeras. (n = 4 mouse-rat chimeras; 6 weeks old).

Table 1 Data regarding the development efficiency of interspecies chimeras

Donor cell lines	Passage number	No. of embryos transplanted	No. of living fetuses (% of transplanted embryos)	No. of chimeric fetuses (% of transplanted embryos)	
C57 GFP mESCs	P17-20	224	55 (24.6)	40 (17.9)	
129 GFP mESCs	P5-10	203	55 (27.1)	29 (14.3)	

Then, we analyzed the chimeric ratio of interspecies chimeras. We first detected the fluorescence signals of chimeras, indicating that mouse ESC lines could contribute to different parenchymal organs (heart, liver, spleen, lung, kidneys, and brain) (Figure 1E). Subsequently, more chimeras were explored by PCR detection of genomic Gapdh, showing evidence of chimerism in the six aforementioned parenchymal organs (Figure 1F). Next, we detected GFP-positive cells derived from various parenchymal organs using fluorescence-activated cell sorting (FACS). The results proved that different proportions of chimerism were present in these parenchymal organs, and the percentage of GFP-positive cells in the chimeric livers varied up to 15%–17% (Figure 1G). This suggests the possibility of obtaining hepatocytes from interspecies chimeras.

Interspecies chimera-derived hepatocytes exhibited mature hepatic function in vitro

Hepatocytes are the primary cell type in the liver, accounting for approximately 60% of the total number of liver cells and 80% of the liver volume (Vekemans and Braet, 2005). We investigated whether interspecies chimera-derived hepatocytes have mature hepatic function. A high percentage of chimerism was observed in some areas of the interspecies chimeric livers in situ (Figure 2A). Immunofluorescence staining showed that chimeric hepatocytes normally expressed the mature hepatic markers, ALB, fumarylacetoacetate hydrolase (FAH), and asialoglycoprotein receptor 1/2 (Figure 2B). The percentage of GFP-positive hepatocytes at low magnification was 44.07%, 34.09%, and 49.42%, respectively (Figure S2A). In addition, staining for the bile duct marker, cytokeratin 19 indicated that murine-derived cells could contribute not only to the liver parenchyma, but also to the bile duct of rats (Figure S2B).Figure 2 Interspecies chimera-derived hepatocytes exhibited mature hepatic function in vitro

(A) Representative fluorescence images showing that GFP mouse ESCs derived from 129 mouse strain contribute to the liver in the 8-week-old mouse-rat chimera. Arrowheads indicate the GFP signals. Top scale bar, 5 mm; bottom scale bar, 1 mm.

(B) Representative immunofluorescence images showing the expression of hepatocyte markers, including ALB, FAH, and ASGR1/2, in the liver tissue of mouse-rat chimeras. Red, ALB, FAH, or ASGR1/2; blue, Hoechst. Scale bar, 50 μm.

(C) Live GFP-positive hepatocytes obtained by flow sorting with DAPI at a rate of up to 20.6%. FITC, fluorescein isothiocyanate.

(D) Lipid accumulation in chimeric hepatocytes (Chimera Hep) analyzed by Nile red staining. Scale bar, 50 μm.

(E) Glycogen storage in chimeric hepatocytes (Chimera Hep) analyzed by periodic acid-Schiff staining. Scale bar, 50 μm.

(F) The capacity for urea synthesis in chimeric hepatocytes (Chimera Hep) is normal. WT hepatocytes (WT Hep) are directly used as a positive control. (n = 3 wells independent replicates per group). Results were the mean ± SEM. n.s., no significance on unpaired 2-tailed t test.

(G) Representative immunofluorescence images showing the expression of ALB in chimeric hepatocytes. Red, ALB; blue, Hoechst. Scale bar, 50 μm.

We then attempted to efficiently and viably isolate hepatocytes from interspecies chimera-derived livers using an approach with a high viability of 95%, as reported previously (Charni-Natan and Goldstein, 2020). We selected adult interspecies chimeras of 8 weeks to isolate hepatocytes. Therefore, it took us 8 weeks from the generation of interspecies chimeras to isolate hepatocytes. After perfusion and digestion of the liver, GFP-positive hepatocytes were enriched using the optimized flow sorting method (Figure S2C). Several interspecies chimeric livers were used for hepatocyte separation and flow sorting, and the percentage of live GFP-positive hepatocytes was up to 20.6% (Figures 2C and S2D). We detected the survival rate of hepatocytes after FACS with cell counter, and the survival rate of hepatocytes after each flow sorting basically was approximately 50%. We then cultured the hepatocytes in vitro and assessed their hepatic functions. Nile red staining showed that chimeric hepatocytes accumulated lipids similar to wild-type (WT) hepatocytes (Figure 2D). The results of periodic acid-Schiff (PAS) staining indicated that chimeric hepatocytes could store glycogen as normally as the WT hepatocytes (Figure 2E). There was no significant difference in urea synthesis between the chimeric hepatocytes and controls (Figure 2F). In addition, chimeric hepatocytes (GFP-positive) cultured in vitro expressed mature hepatic marker, ALB normally (Figure 2G). These results indicate that interspecies chimera-derived hepatocytes exhibit mature hepatic function in vitro.

Gene expression profiles indicated that interspecies chimera-derived hepatocytes were more similar to endogenous hepatocytes

We used RNA sequencing (RNA-seq) to analyze gene expression in interspecies chimera-derived hepatocytes (Figure 3A). Heatmap analysis revealed a remarkable concordance in the expression of typical liver genes between interspecies chimera-derived hepatocytes and WT hepatocytes (Figures 3B and S3A). To distinguish whether the hepatocytes were originated from mice or rats, differential analysis through sample sequence matching indicated that the sorted GFP-positive hepatocytes were indisputably from mouse livers rather than rat livers (Figures 3C and S3B).Figure 3 Gene expression profiles indicated that interspecies chimera-derived hepatocytes were more similar to endogenous hepatocytes

(A) Flow chart for interspecies chimera-derived hepatocytes obtained by in situ collagenase perfusion and flow sorting and gene expression profiling of interspecies chimera-derived hepatocytes by RNA-seq.

(B) Heatmap of expression levels of hepatocyte marker genes in chimeric hepatocytes (Chimera), mouse WT hepatocytes (WT) as positive controls, and mouse embryonic fibroblast (MEF) as negative controls. For the full list, see Figure S3A.

(C) Heatmap of expression levels of hepatocyte marker genes among mouse WT hepatocytes (WT), chimeric hepatocytes (Chimera), and SD rat WT hepatocytes (SD) for comparison, which proves chimeric hepatocytes belong fully to murine origin. For the full list, see Figure S3B.

(D) Hierarchical clustering analysis of the gene expression of mouse WT hepatocytes (WT), chimeric hepatocytes (Chimera), iHep cells (iHep), in vitro differentiated hepatocytes (IV diff), and WT hepatocytes reported (WT Ref.).

(E) Heatmap of expression levels of hepatocyte marker genes among chimeric hepatocytes, iHep cells, and in vitro differentiated hepatocytes reported for comparison. WT hepatocytes are used as positive controls. For the full list, see Figure S3C.

(F) Enriched GO terms of upregulated biological processes involved in chimeric hepatocytes and WT hepatocytes compared with iHep cells and in vitro differentiated hepatocytes.

(G) Enriched GO terms of downregulated biological processes involved in chimeric hepatocytes and WT hepatocytes compared with iHep cells and in vitro differentiated hepatocytes.

(H) Enriched KEGG pathways of upregulated signaling pathways involved in chimeric hepatocytes and WT hepatocytes compared with iHep cells and in vitro differentiated hepatocytes.

(I) Enriched KEGG pathways of downregulated signaling pathways involved in chimeric hepatocytes and WT hepatocytes compared with iHep cells and in vitro differentiated hepatocytes.

We performed an extensive investigation to determine whether hepatocytes derived from interspecies chimeras closely resembled those naturally developed in vivo or hepatocytes derived in vitro. Cluster analysis revealed that chimeric hepatocytes displayed greater similarity to WT hepatocytes than to induced hepatocyte-like (iHep) cells derived from mouse embryonic fibroblasts (MEFs) reprogramming (Horisawa et al., 2020), as well as hepatocytes generated through hepatoblast differentiation in vitro (Belicova et al., 2021) (Figure 3D). Differential analysis revealed that interspecies chimera-derived hepatocytes expressed hepatic genes, as well as an array of genes associated with hepatic functions, including cytochrome P450 activity, glucose metabolism, lipid metabolism, complement activation, and urea cycle (Figures 3E and S3C–S3H). This comprehensive expression pattern underscores the remarkable capacity of interspecies chimera-derived hepatocytes to perform a multitude of hepatic functions. The Gene Ontology (GO) analysis revealed that the expression of genes related to hepatic functions, such as lipid metabolic process, steroid metabolic process, and liver development, was significantly upregulated in interspecies chimera-derived hepatocytes (Figure 3F). Furthermore, our interspecies chimera-derived hepatocytes exhibited lower expression of crucial biological processes, such as apoptosis, cell death, aging, positive regulation of hepatic stellate cell (HSC) activation, and immune inflammation, than both iHep cells and in vitro differentiated hepatocytes (Figure 3G). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes indicated that interspecies chimera-derived hepatocytes exhibited significant upregulation of pathways associated with hepatic functions, such as fatty acid degradation and steroid hormone biosynthesis (Figure 3H). Conversely, pathways related to hepatocellular carcinoma, cell senescence, hepatitis B, immune inflammation, and apoptosis were notably downregulated in chimeric hepatocytes (Figure 3I). These results provide strong evidence to support the notion that interspecies chimera-derived hepatocytes bear a striking resemblance to endogenous hepatocytes. They not only exhibit a remarkable similarity in their molecular profiles, but also display a more comprehensive range of hepatic functions.

Mouse chronic liver fibrosis was alleviated after interspecies chimera-derived hepatocyte transplantation

Finally, we explored whether interspecies chimera-derived hepatocytes could alleviate fibrosis caused by liver injury to determine their functions in vivo. We used carbon tetrachloride (CCl4) to model chronic fibrosis injury in immunodeficient mice (NOD SCID) for 6 weeks, followed by HT (Figure 4A). Serum samples examination showed a significant increase in both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities after fibrosis modeling, indicating liver injury (Figure 4B). The liver surface contours of the CCl4-induced chronic liver fibrosis model were irregular, unsmooth, and relatively rough, with jagged edges (Figure 4C). Hematoxylin and eosin (H&E) staining indicated ballooning degeneration and the presence of apoptotic cells in CCl4-induced chronic liver fibrosis models (Figure S4A). Masson trichrome staining revealed significant collagen deposition after long-term CCl4-induced injury, indicating typical fibrosis (Figure S4B). This evidence demonstrates that the model of chronic liver fibrosis has been successfully established.Figure 4 Mouse chronic liver fibrosis was alleviated after interspecies chimera-derived hepatocyte transplantation

(A) Preparation of mice with chronic fibrotic liver injury and chimeric hepatocyte transplantation. CCl4 is used to model chronic fibrosis injury in NOD SCID mice with a corn oil injection group as concurrent controls. At the end of 6 weeks of CCl4 injection, interspecies chimera-derived hepatocytes are injected into the spleens of chronic liver fibrosis models, with WT hepatocytes injected as a positive control and PBS injected as a negative control.

(B) ALT and AST activities in CCl4-treated mice measured and normalized to the values from NOD SCID mice injected with corn oil before and after fibrosis modeling. (n = 3 mice in each group at each time point). Results were the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and n.s., no significance on unpaired 2-tailed t test.

(C) Typical liver images with (CCl4) or without (corn oil) CCl4 treatment.

(D) Typical liver images after PBS (w/PBS) or WT hepatocytes (w/WT Hep) or chimera hepatocytes (w/Chimera Hep) injection on day 7 in CCl4-treated mice compared with NOD SCID mice injected with corn oil (corn oil). Arrowheads indicate GFP signals. Scale bar, left 1 cm, right 0.5 mm.

(E) ALT and AST activities in PBS-transplanted (w/PBS), WT Hep-transplanted (w/WT Hep), and Chimera Hep-transplanted (w/Chimera Hep) mice measured and normalized to the values of NOD SCID mice injected with corn oil (corn oil) on day 7 after injection. (n = 3 mice in each group). Results were the mean ± SEM. ∗p < 0.05 on unpaired 2-tailed t test.

(F) Pathological section of H&E staining on day 7 after injection. Arrowheads indicate ballooning degeneration and the presence of apoptotic cells. Scale bar, 100 μm.

(G) Pathological section of Masson staining on day 7 after injection. Arrowheads indicate remarkably increased collagen fibers. Scale bar, 100 μm.

(H) GFP hepatocyte localization of representative immunofluorescence images on day 7 after injection. Red, ALB; blue, Hoechst. Scale bar, left 100 μm, right 50 μm.

Observation of the liver on day 7 after transplantation indicated that the smoothness of the liver surface was improved in both the interspecies chimeric HT group and the WT HT group and that the transplanted hepatocytes could exist in the host liver (Figure 4D). Similar to the WT HT group, ALT and AST activity decreased significantly after chimeric HT, demonstrating that liver injury was alleviated (Figure 4E). H&E staining revealed that the chimeric HT group was similar to the WT HT group, with a significant decrease in the number of necrotic hepatocytes (Figure 4F). Masson trichrome staining also proved that collagen deposition was significantly reduced after interspecies chimera-derived HT, similar to WT HT, demonstrating the alleviation of tissue fibrosis (Figure 4G). Immunofluorescence staining for cellular localization showed that chimeric hepatocytes exhibited a certain degree of colonization in the host liver, similar to WT hepatocytes (Figure 4H). The range of engraftment efficiency of transplanted interspecies chimera-derived hepatocytes was between 3.43% and 15.92%, and the average engraftment efficiency of transplanted interspecies chimera-derived hepatocytes was 8.74% (Figure S4C). These results indicate that interspecies chimera-derived hepatocytes exhibit functionality, which may provide an alternative source of hepatocytes for transplantation to alleviate chronic liver fibrosis caused by long-term liver injury.

Discussion

In this study, we isolated mouse ESC-derived hepatocytes from mouse-rat chimeras via blastocyst complementation, performed hepatic function identification, and explored their ability to contribute to the recovery of liver diseases. Our results indicated that interspecies chimera-derived hepatocytes had mature hepatic function and were more similar to endogenous hepatocytes. More importantly, they were capable of alleviating chronic liver fibrosis caused by long-term liver injury in mice. This study demonstrates the potential of chimeric hepatocytes as a reliable and effective source of hepatocytes for future clinical applications.

HT is a potential alternative to LT, and hepatocytes from interspecies chimeras via blastocyst complementation can be an alternative source when there is a shortage of primary hepatocytes. Through intraspecies blastocyst complementation in mice, full repopulation of donor cells from PSCs was observed in the livers of recipients, and donor cell-derived hepatocytes regenerated the liver after transplantation, which expanded the source of hepatocytes (Espejel et al., 2010). Comprehensive evaluation of hepatic functions and therapeutic potential after transplantation of interspecies chimera-derived hepatocytes developed through IBC will hold great significance for prospective clinical application. Our study represents detailed functional identification and evaluation of hepatocytes generated through IBC, including hepatic function exploration and therapeutic function after transplantation, which provides an additional source of hepatocytes and therapeutic method of HT for clinical application.

In this study, we demonstrated that interspecies chimera-derived hepatocytes retained some of the functional characteristics of mature hepatocytes. Moreover, bioinformatics analyses indicated that early chimeric ESCs, participated in a series of developmental processes in vivo, were closer to endogenous hepatocytes and had more comprehensive functions than iHep cells and differentiated hepatocytes in vitro. These results also indicated that IBC with donor PSCs could provide all the conditions and factors required for hepatocyte and liver development because differentiation was appropriately induced in vivo and signal recognition and communication between cells were more adequate. Compared to the GO analysis results on our interspecies chimera-derived hepatocytes, gene expression in iHep cells and differentiated hepatocytes in vitro was significantly upregulated in hepatic stellate cell activation. This represented hepatic fibrosis since HSCs could secrete large amounts of extracellular matrix, especially collagen α 1 (I), resulting in hepatocyte damage (Kisseleva and Brenner, 2007). In addition, interspecies chimera-derived hepatocytes relieved chronic liver fibrosis caused by CCl4, indicating that they could play a role in vivo in future clinical transplantation applications. Moreover, we found that our interspecies chimera-derived hepatocytes contributed to the bile duct of the recipient, which was consistent with the observations of a previous study (Kobayashi et al., 2010). This result indicated that other types of cells besides hepatocytes could be generated in the liver via IBC, proving that IBC had the potential to help donor cells contribute to all types of cells in the liver and promote reconstruction of the whole liver (Aravalli, 2021). Furthermore, compared with hepatocellular carcinoma cells (Zhou et al., 2023), interspecies chimera-derived hepatocytes exhibited high expression of the tumor suppressor gene Trp53 and low expression of Kras and Pik3ca and vascular endothelial growth factor genes (Figure S3I), and tumors were not observed in the liver on day 7 after HT. These results proved that interspecies chimera-derived hepatocytes did not produce tumors.

Given the current shortage of donors for LT and HT, it is more promising to obtain human hepatocytes or livers from large animals, such as pigs, by IBC, as this yields higher numbers of hepatocytes and provides support for the clinical application. However, owing to some barriers such as variations between individuals in interspecies chimeras and the randomness of chimerism, the clinical application of IBC remains limited (Wu et al., 2016). The approach referring to recipient blastocysts with genetic mutations disabling organogenesis and transplantation of WT donor PSCs, which results that the donor cells fill the induced competitive niche and form the missing organ as chimeric embryos develop (Larson et al., 2021), can be a promising way to achieve constant and high chimeric efficiency in the liver. Defective niches in the liver obtained by gene modification such as FAH deficiency demonstrated that chimeric livers could be generated in which all hepatocytes were derived from donor cells (Espejel et al., 2010). Building on our research involving interspecies chimerism between mice and rats, we aim to further explore the possibility of acquiring functional primate-derived livers or hepatocytes from large animal models.

In addition, we observed the ability of colonization and alleviating chronic fibrosis of interspecies chimera-derived hepatocytes at the early stage after transplantation, but not long-term observation. Previous report indicated that rat mature hepatocytes could survive for more than 4 months after transplanted into fibrotic rat livers and proliferate competitively compared to host hepatocytes (Yovchev et al., 2014). Considering that the interspecies chimeric HT group exhibited similar therapeutic effects to the WT HT group, and the chimeric hepatocytes exhibited a certain degree of colonization in the host liver similar to WT hepatocytes, we assume that transplanted interspecies chimera-derived hepatocytes can effectively repopulate the fibrotic livers and produce a long-term curative effect. Moreover, CCl4 is currently the most widely used induction drug in the establishment of animal models of liver fibrosis and cirrhosis (Du et al., 2022). Since inborn error of metabolism models such as Fah−/− mice or acute liver failure models are widely used for in vivo validation, we will also focus on the application of interspecies chimera-derived hepatocytes in other models in the future. Furthermore, our research involved the use of CCl4-induced chronic liver fibrosis, which might exhibit some degree of regression after the withdrawal of CCl4 (Cui et al., 2013). This could potentially be a contributing factor to the alleviation of chronic liver fibrosis in mice.

In conclusion, we isolated interspecies chimera-derived hepatocytes and evaluated their functions from multiple perspectives, in vitro and in vivo. The results showed that interspecies chimera-derived hepatocytes had mature hepatic function, were more similar to endogenous hepatocytes than hepatocytes derived in vitro, and could mitigate chronic liver fibrosis. Our results suggest that not only interspecies chimera-derived hepatocytes can be a reliable source of hepatocytes, but also, in principle, interspecies chimera-derived HT can be an effective therapeutic method for HT.

Experimental procedures

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Wei Li (liwei@ioz.ac.cn).

Materials availability

This study did not generate new unique reagents. All unique/stable reagents generated in this study are available from the lead contact without restriction. All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics, 2021) (Chen et al., 2021) in National Genomics Data Center (Nucleic Acids Res, 2022) (Members and Partners, 2021), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA012646) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

Experimental animals

Mice were housed in the animal care facility of the Institute of Zoology, Chinese Academy of Sciences, according to the institutional guidelines for the care and use of laboratory animals. 129-Gt(ROSA)26Sortm1(CAG−EGFP)Luo/J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Sprague-Dawley (SD) rats, C57 strain mice, and NOD SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).

Model system and permissions

All animal research and care procedures were approved by the Institutional Animal Use Committee of the Institute of Zoology at the Chinese Academy of Sciences, Beijing, China (Approval number: IOZ-IACUC-2021-096).

Mouse ESC derivation and cell culture

The 129-strain GFP mouse ESC line was derived from the blastocysts from crossbreeding of the male and female mice of 129-Gt(ROSA)26Sortm1(CAG−EGFP)Luo/J strain using standard procedures (Bryja et al., 2006). The C57 strain GFP mouse ESC line was established from blastocysts of C57 strain mice and labeled with GFP using the PiggyBac system. After derivation, the cells were cultured in 2iL medium composed of N2B27 medium (detailed components are listed in Table S1), 3 μM CHIR99021 (04-0004; Stemgent, Cambridge, MA, USA), 1 μM PD0325901 (04–0006; Stemgent, Cambridge, MA, USA), and 1,000 U/mL mLIF (ESG1107; Millipore, Burlington, MA, USA) on MEF cells with mitomycin-c-treated, namely feeder cells. Mouse ESCs were passaged every 4–5 days at a split ratio of 1:20.

Preparation of rat blastocysts and blastocyst injection

Blastocysts were obtained by mating male SD strain rats with female SD strain rats. The blastocysts were gently flushed out from the uteri of 4.5 days post coitus (dpc) pregnant SD female rats with HEPES-buffered mR1ECM medium (270 mOsm) (Li et al., 2017). The harvested blastocysts were transferred into mR1ECM medium (246 mOsm) (Oh et al., 1998) and cultured in 5% CO2 at 37°C for 30 min, when the blastocoele of the blastocysts was apparent.

For micromanipulation, mouse ESCs were trypsinized into individual cells and suspended in M2 medium (M7167; Sigma-Aldrich Corp., St. Louis, MO, USA). Embryo injections were performed with a micromanipulation system equipped with a manual manipulator (MMO-202; Narishige, Amityville, NY, USA), a piezo drive system (PMM-150; Prime Tech, Ibaraki, Japan), and an inverted microscope with differential interference contrast optical components (DMI-3000B; Leica, Wetzlar, Germany), as described previously (Li et al., 2017). About 8–10 mouse ESCs were injected into a rat blastocyst cavity near the inner cell mass, and after being cultured in mR1ECM for 30 min, the chimeric embryos were transferred into the uteri of 3.5-dpc pseudo-pregnant SD recipient rats. Images of reconstructed blastocysts were captured using an inverted microscope (DMI-8; Leica, Wetzlar, Germany).

Genotyping

Genotyping analysis was performed by PCR detection of genomic Gapdh, with mouse Gapdh (mGapdh) and rat Gapdh (rGapdh) as controls, as reported previously, with some modifications (Isotani et al., 2011). Genomic DNA was extracted from each sample using the E.Z.N.A. MicroElute Genomic DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA). Ex Taq (RR001A; Takara, Shiga, Japan) was used for the PCR. Primer pair sequences are listed in Table S2. The DNA fragments were amplified for 40 cycles under the following conditions: 94°C for 30 s, 62°C for 30 s, and 68°C for 30 s.

Proportions of chimerism analysis by FACS

Chimeras were sacrificed, and various parenchymal organs (heart, liver, spleen, lung, kidneys, and brain) were removed separately. After being split into fragments in Petri dishes, organs were digested into single cells by collagenase IV (17104019; Gibco, Billings, MT, USA) and trypsin-EDTA (0.25%) (25200072; Gibco, Billings, MT, USA) at 37°C. Digestion was stopped using Dulbecco’s modified Eagle’s medium (C11995500BT; Gibco, Billings, MT, USA) supplemented with 10% FBS (10099141C; Gibco, Billings, MT, USA). The chimeric ratio was assessed based on GFP fluorescence using FACS analysis. FACS was performed and data were recorded using a BD FACS Aria II (BD Biosciences, Franklin Lakes, NJ, USA). Then, we analyzed the proportion of GFP-positive cells in mouse-rat chimeras and graphed them using FlowJo software (FlowJo, Ashland, OR, USA).

Immunofluorescence staining

For immunofluorescence staining, hepatocytes cultured overnight were fixed with 4% paraformaldehyde (PFA) (DF0135; LEAGENE, Beijing, China) for 15 min and subsequently permeabilized and blocked with 0.5% Triton X-100 (T8787; Sigma-Aldrich Corp., St. Louis, MO, USA) and 2% bovine serum albumin (BSA) for 1 h. Then, hepatocytes were incubated in primary antibody overnight at 4°C and secondary antibody at room temperature in the dark for 1 h. The DNA was stained with Hoechst 33342 (H3570; Invitrogen, Waltham, MA, USA) for 10 min. Images were captured using a two-photon confocal laser-scanning microscope (TCS Sp8; Leica, Wetzlar, Germany).

For immunofluorescence staining of liver tissues, to avoid the subsequent influence of blood in the liver on immunofluorescence staining, the livers were first infused with phosphate-buffered saline (PBS) (21-040-CVR; Corning, NY, USA) and then removed to observe the fluorescence under a microscope. After fixation with 4% PFA and graded dehydration, the livers were frozen in liquid nitrogen with OCT (4583; Sakura, Torrance, CA, USA) and then sliced into 10-mm sections. The frozen sections were incubated for 30 min at room temperature. After rinsing with PBS three times, the slices were incubated in blocking buffer (5% BSA with 0.5% Triton X-100) for 1 h and subsequently incubated with the primary antibody overnight at 4°C. After washing three times with PBS, the slices were incubated with secondary antibody for 1 h at room temperature in the dark. The DNA was stained with Hoechst 33342 (H3570; Invitrogen, Waltham, MA, USA) for 10 min. Images were captured using a two-photon confocal laser scanning microscope (TCS Sp8; Leica, Wetzlar, Germany) (for the detection of chimeric liver) or PerkinElmer Launches Vectra Polaris Automated Quantitative Pathology Imaging System (PerkinElmer, Waltham, MA, USA) (for 7 days post-HT to ascertain liver injury repair).

The primary antibodies were Anti-GFP (1:200, Abcam, Ab13970), Anti-ALB (1:50, Santa Cruz, Sc-271605), Anti-FAH (1:200, Abcam, Ab151998), Anti-ASGPR1/2 (1:50, Santa Cruz, Sc-166633), and Anti-CK19 (1:100, Invitrogen, MA1-82041). Goat anti-chicken IgG 488 (1:500, Invitrogen, A11039), Donkey anti-mouse IgG 546 (1:500, Invitrogen, A10036), and Donkey anti-rabbit IgG 594 (1:500, Invitrogen, A21207) were used as secondary antibodies.

Isolation of adult hepatocytes

Adult hepatocytes were isolated as previously reported (Charni-Natan and Goldstein, 2020). Live GFP hepatocytes were stained with 4ʹ, 6-diamidino-2-phenylindole (D1306; Invitrogen, Waltham, MA, USA) by flow sorting (MoFlo XDP; Beckman, Brea, CA, USA) under low-pressure and low-speed conditions. An adult rat has about 1.5 × 108 total hepatocytes. Since the chimerism ratio in the liver of our interspecies chimeras was 15%–17% (Figure 1G), the theoretical number of mouse hepatocytes isolated from an interspecies chimera is about 2.25 × 107–2.55 × 107. In our experiment, considering the flow sorting time, flow sorting speed, and hepatocyte viability after flow sorting, we sorted 5 × 106 GFP-positive hepatocytes from an interspecies chimera for hepatic function detection and transplantation therapy.

Culture of hepatocytes

Hepatocytes were plated at a density of 3 × 104 cells/well in 96-well plates coated with collagen I (354236; Corning, NY, USA) and cultured overnight in complete culture medium of mouse hepatocytes (CM-M033; Procell, Wuhan, China).

Assays for Nile red staining, PAS staining, and urea synthesis

For Nile red staining, hepatocytes were washed twice with PBS, fixed in 4% PFA for 10 min, followed by Nile red staining in 1 μM working solution (72485-100 mg; Sigma-Aldrich Corp., St. Louis, MO, USA) for 30 min in the dark, and then washed twice by PBS. The DNA was stained with Hoechst 33342 (H3570; Invitrogen, Waltham, MA, USA) for 5 min, and an anti-fluorescence quencher was added to the cells and observed under a fluorescence microscope.

Hepatocytes were stained using a glycogen PAS stain kit (G1360; Solarbio, Beijing, China) following the manufacturer’s instructions.

To determine urea secretion, the supernatants of the hepatocyte cultures were collected from 96-well plates after 24-h culture. The concentration of secreted urea was measured using a urea assay kit (DIUR-100; Bioassay Systems, Hayward, CA, USA), according to the manufacturer’s instructions.

RNA-seq library preparation and data analysis

One hundred thousand live hepatocytes were obtained by flow cytometry from mouse (with GFP), rat (without GFP), chimera-1 (with GFP), and chimera-2 (with GFP) groups to build an RNA-seq library (two replicates per group). After centrifugation at 50 × g, total RNA was extracted from the hepatocytes using TRIzol reagent (15596018; Thermo Fisher Scientific, Waltham, MA, USA). The Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) was used for the RNA-seq analysis.

FastQC (v0.11.9; Babraham Bioinformatics) was used to evaluate the raw quality of the paired-end reads. Rattus norvegicus and mouse reference genomes (mm10) were used for genomic alignment, which was performed using STAR (version 2.7.7a) aligner. The read count for each gene was determined using StringTie (version 2.1.1). The Deseq2 R package (version 1.30.1), using |Log2-fold change| > 1 and p value < 0.05 as thresholds, was used to identify genes that differed in expression between samples. With the help of the clusterProfiler (3.14.3) and org.Mm.eg.db (3.10.0) packages, the analysis of differentially expressed genes was carried out using GO pathway analysis and KEGG pathway analysis. We used the pheatmap package (1.0.12) and hclust package to perform unsupervised hierarchical clustering analysis. Pheatmap (1.0.12) was used to display the heatmap. R programs, such as tidyr (1.1.2), were used to perform principal component analysis. Published RNA-seq data were downloaded from the NCBI GEO GSE210709 (MEF) (Bai et al., 2022) and GSE176069 (WT Ref. and IV diff) (Belicova et al., 2021), and GSE90534 iHep datasets (Horisawa et al., 2020).

Animal treatment and hepatocytes transplantation

CCl4 (289116-100mL; Sigma-Aldrich Corp., St. Louis, MO, USA) was used to induce chronic liver fibrosis. The CCl4 was diluted with corn oil (C8267-500mL; Sigma-Aldrich Corp., St. Louis, MO, USA) to a final concentration of 20%. CCl4 (2.8 μL/g) was injected intraperitoneally into 8-week-old female NOD SCID mice twice a week for 6 weeks, and corn oil was injected into a control group. At the end of 6 weeks of CCl4 injection, 5 × 105 hepatocytes (WT hepatocytes or interspecies chimera-derived hepatocytes) were injected into the spleens of the chronic liver fibrosis models, and PBS was injected as a negative control. Seven days after HT, all mice were sacrificed for analysis.

Liver injury and repair tests

During CCl4 injection, whole blood was suctioned periodically via capillaries from the inner canthus vein, and plasma was collected after allowing the blood to stand at room temperature for 2 h, followed by centrifugation at 3,000 rpm for 30 min at 4°C. ALT and AST were determined by Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). Six weeks after CCl4 injection, the liver was removed and paraffin-embedded for histological analysis, including H&E and Masson staining. All tests were also conducted at the end of an additional 7 days post-HT to ascertain liver injury repair.

Statistical analysis

For statistical analysis, the unpaired 2-tailed t test was performed using GraphPad Prism 8 software. All of the data were presented as mean ± SEM. p values < 0.05 were considered statistically significant. In all figures, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and n.s., no significance.

Supplemental information

Document S1. Figures S1–S4 and Tables S1–S3

Document S2. Article plus supplemental information

Acknowledgments

This study was supported by grants from the 10.13039/501100012166 National Key Research and Development Program (2019YFA0110102 and 2021YFA0719303 to T.L. and 2018YFE0201100 to L.W.), the CAS Project for Young Scientists in Basic Research (YSBR-012 to W.L.), the 10.13039/501100001809 National Natural Science Foundation of China (32225030 to W.L., 31621004 to Q.Z. and W.L., and 32071456 and 81972633 to Y.Z.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030400 to W.L.), the international cooperation project of China Manned Space Program, and the International Partnership Program of Chinese Academy of Sciences (no. 152111KYSB20160004 to Q.Z.).

The authors thank W.J., P.L., M.G., and W.K. for their help with animal studies and embryonic micromanipulation experiments, Z.L. and J.Y. for their help in the identification of related molecular biology experiments, and J.J., Q.M., T.L., and X.Y. for their help with the FACS experiments. We appreciate the help of S.L. and H.Q. in the confocal imaging and panoramic histocyte systems.

Author contributions

Q.Z., W.L., T.L., G.F., and X.L. conceived the idea and designed the study; Q.Z., W.L., and T.L. supervised the project; C.H., H.J., L.J., J.L., J.X., and L.W. performed the experiments; J.D. analyzed the sequencing data; T.C. was involved in the methodology; C.H., H.J., J.D., and T.L. wrote the manuscript; and Y.Z. was involved in the manuscript preparation and helped revise the manuscript. All authors read and approved the final version of the manuscript.

Declaration of interests

The authors declare no competing interests.

Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2024.04.006.
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