There is an enormous clinical need for liver transplant tissue. Bioengineered livers might ultimately be used as a bridge to or alternative for whole organ transplantation. In new work, Stevens et al. fabricated human artificial liver “seeds” in biomaterials that were able to grow and expand after implantation into mice in response to liver injury. After growth, the human artificial liver seeds were able to carry out normal liver functions such as production of human proteins like transferrin and albumin. This study suggests that implanted engineered tissue seeds should be able to expand in response to the body’s own repair signals.

Control of both tissue architecture and scale is a fundamental translational roadblock in tissue engineering. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. We fabricated a structurally organized engineered tissue unit that expanded in response to regenerative cues after implantation into mice with liver injury. Specifically, we found that tissues containing patterned human primary hepatocytes, endothelial cells, and stromal cells in a degradable hydrogel expanded more than 50-fold over the course of 11 weeks in mice with injured livers. There was a concomitant increase in graft function as indicated by the production of multiple human liver proteins. Histologically, we observed the emergence of characteristic liver stereotypical microstructures mediated by coordinated growth of hepatocytes in close juxtaposition with a perfused vasculature. We demonstrated the utility of this system for probing the impact of multicellular geometric architecture on tissue expansion in response to liver injury. This approach is a hybrid strategy that harnesses both biology and engineering to more efficiently deploy a limited cell mass after implantation.

Here, we fabricated a liver tissue seed that could support the in situ expansion of its cellular components in response to systemic regenerative cues after implantation in mice with liver injury. Our efforts were informed by reports of the importance of paracrine signaling between hepatocytes, endothelial cells, and stromal cells in both liver development and regeneration processes ( 6 – 9 ), as well as by our prior engineering efforts that demonstrated context-dependent cell signaling in microfabricated tissue microenvironments using a range of biomaterials ( 2 , 19 – 23 ). Our combined approach yielded an engineered, fully human tissue seed composed of human endothelial cells, hepatocytes, and fibroblasts in a degradable hydrogel that engrafted ectopically in mice and expanded more than 50-fold in situ in response to regenerative signals. The resultant human organoid phenocopied several aspects of native liver structure and function, including perfused vascular networks, self-assembled structures resembling bile ducts, and production of human liver proteins secreted into mouse blood.

A different approach may be to nucleate a “seed” of an organ derived from mature cell populations that can grow by in situ expansion, as seen both in hepatic embryogenesis wherein primordial tissue buds grow into vascularized organs and in regenerative adult responses wherein hepatocytes and their vasculature undergo coordinated expansion ( 6 – 9 ). As a step toward this goal, cells have been expanded after engraftment in solid organs by manipulating cell signaling pathways ( 10 – 12 ) or by creating a repopulation advantage for graft cells using injury models ( 13 – 18 ). However, each of these methods depends on the ability of grafted cells to self-organize to form larger, organized tissue structures and leaves open the question as to the role of controlled multicellular architectural interactions during the expansion and ultimate function of grafted tissues.

A variety of approaches have been examined to promote vascularization and architectural structural control of solid engineered tissues. For example, inclusion of randomly organized or patterned endothelial cells has improved both the engraftment rate and persistence of metabolic tissues ( 6 – 8 ). Decellularization of native structures ( 9 , 10 ) and biofabrication techniques, such as microtissue molding ( 1 , 2 ) and bioprinting ( 3 – 5 ), have provided further ways to organize cells into scalable vascularized architectures, but all of these approaches offer limited utility for large solid organs because tissues must be perfused rapidly (within ~1 hour of tissue assembly) to prevent ischemic injury.

Advances in tissue engineering have enabled the generation of numerous tissue types that can recapitulate many aspects of native organs, bringing closer the promise that engineered tissues may ultimately replace whole-organ transplantation ( 1 , 2 ). However, constructing complex solid organs remains a major physical and biological challenge. For engineered tissues that can be fed by diffusion of nutrients from the environment (such as the cornea and skin), thick tissues with low metabolic requirements (such as cartilage), or small-scale endocrine tissues (such as β cells of the pancreas), the recapitulation of complex tissue architecture has not been a limiting factor for clinical translation ( 1 , 2 ). However, providing structural organization is likely important for large, metabolically active solid organs such as the heart, kidney, and liver. For example, the liver contains more than 100 billion hepatocytes, all positioned within 50 μm of the circulation ( 3 ). The organization of the circulation and its lining endothelium are integral aspects of the functional organ and are critical to the delivery of vital nutrients to the entire parenchyma of tissue, as well as to the cell-cell interactions that define juxtacrine and paracrine signals that drive diverse processes such as embryological development, organ function, and regeneration ( 4 , 5 ).

RESULTS

Construction of human liver tissue seeds from cellular components We first sought to create an engineered “tissue seed” candidate by arranging a combination of human cell types in a format that would allow for expansion of the tissue seeds in situ in response to regenerative cues. Previous work had demonstrated the importance of paracrine signals between hepatocytes, endothelial cells, and stromal cells (6–9) in regeneration, as well as the requirement for defined spatial organization of engineered tissues for optimizing cellular function (1, 2, 21). We therefore incorporated human hepatocytes, endothelial cells, and stromal cells in structurally organized tissue seeds. To do this, we first used microwell technology to create aggregates of a defined size, composed of combinations of human hepatocytes and normal human dermal fibroblasts (NHDFs; Fig. 1A) (2). Similarly, microtissue molding was used to create patterned endothelial cord structures (1) from human umbilical vein endothelial cells (HUVECs) (Fig. 1B). This fabrication process was adaptable for scaled construction of larger tissues using a bioprinting process that we developed previously (fig. S1) (4). Finally, we coencapsulated the hepatic cellular aggregates with the endothelial cell cords in a fibrin hydrogel to create tissue seeds that were suitable for ectopic implantation in the intraperitoneal mesenteric fat of mice (Fig. 1B). Fig. 1. Construction of human liver seed grafts. (A) Human hepatic aggregates containing human primary hepatocytes and NHDFs were created using pyramidal microwells. (B) Hepatic aggregates (red) were then combined with geometrically patterned human endothelial cell cords (green) in a fibrin hydrogel to create “liver tissue seeds” that were then implanted ectopically into FNRG mice. Right: Blue-gloved finger demonstrates macroscopic scale of human liver tissue seeds. (C) Human albumin production by human hepatocytes was enhanced sixfold in liver seed grafts containing both hepatocytes (Hep) and NHDFs compared to seed grafts containing only hepatocytes after culture in vitro for 6 days (***P < 0.0001). (D) Albumin promoter activity was enhanced in implanted liver seed grafts composed of human hepatocytes and NHDFs compared to those containing only human hepatocytes, 6 days after implantation into FNRG mice (**P < 0.01). ROI, region of interest. Scale bars, 400 μm. As an initial characterization step, short-term in vitro tests showed that the addition of NHDFs to hepatocyte aggregates enhanced sixfold the production of human albumin, a measure of hepatic function, in a dose-dependent manner compared to aggregates containing only hepatocytes before encapsulation with endothelial cords (Fig. 1C and fig. S2). Similarly, the albumin promoter activity (21) of tissue seeds containing aggregates of both hepatocytes and NHDFs was enhanced more than eightfold compared to seeds containing hepatocyte-only aggregates 6 days after implantation into the mesenteric fat of healthy nude mice (Fig. 1D). Thus, the combination of human hepatocytes, endothelial cells, and stromal cells yielded candidate human liver tissue seeds that could survive engraftment in vivo.

Expansion of human hepatocytes in ectopic implants after liver injury in host mice We hypothesized that inducing host soluble regenerative signals in mice receiving ectopic implants of liver tissue seeds might trigger these seeds to grow. Accordingly, we implanted human liver tissue seeds into the mesenteric fat of FNRG mice, an immunodeficient mouse model of the liver disease hereditary tyrosinemia type I. FNRG mice were generated by backcrossing fumarylacetoacetate hydrolase–deficient (Fah−/−) mice with nonobese diabetic (NOD) mice, recombinase activating gene–deficient (Rag1−/−) mice, and interleukin-2 receptor γ chain–deficient (Il2rγ-null) mice (13, 24, 25). This mouse strain experiences progressive liver failure unless treated with the small molecule 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). NTBC was administered continuously to FNRG mice in the control group (Fig. 2A) or was given intermittently to induce cycles of liver injury and regeneration. Animals were sacrificed, and grafts were retrieved at 80 days after tissue seed implantation. Grafts were located in the mesenteric fat pad using the suture as a landmark. Fig. 2. Human liver tissue seed grafts expand after host liver injury. (A) Human liver tissue seed grafts were implanted onto the mesenteric fat of FNRG mice. The mice were treated intermittently with the small molecule NTBC to induce liver injury and regeneration or received NTBC continuously and remained injury free. (B) Immunostaining of liver tissue seed grafts explanted 80 days after implantation showed positive staining for the markers cytokeratin-18 and arginase-1 in animals with and without liver injury. Scale bars, 100 μm. (C) Histomorphometry revealed greater cytokeratin-18–positive graft area and volume in animals with liver injury compared to controls. (D) Many Ki67 and cytokeratin-18 double-positive cells were observed in the liver seed grafts from mice with liver injury (left, white arrows); rare Ki67 and cytokeratin-18 double-positive cells undergoing mitosis were also observed (right, white arrows). Graph shows that liver seed grafts in animals with liver injury exhibited a greater number of Ki67 and cytokeratin-18 double-positive cells compared to uninjured animals. Scale bars, 10 μm. *P < 0.05, **P < 0.01 (Student’s t test). To determine whether ectopic liver tissue seeds had expanded in animals in response to chronic injury and regeneration cycles induced by intermittent NTBC dosing compared to control animals, we identified human hepatocytes by immunostaining for human cytokeratin-18, an intermediate filament expressed by hepatocytes, and arginase-1, an enzyme that catalyzes the hydrolysis of arginine to ornithine and urea (26, 27). Grafts from control animals treated continuously with NTBC had small cellular aggregates containing cytokeratin-18– and arginase-1–positive cells dispersed within the hydrogel (Fig. 2B, left). In animals that underwent cycles of injury and regeneration caused by intermittent treatment with NTBC, larger hepatic grafts composed of densely packed cytokeratin-18– and arginase-1–positive cells were observed (Fig. 2B, right). The cytokeratin-18–positive surface area in tissue seed grafts was quantified by a blinded observer using morphometric analysis in histologic sections. Hepatic tissue seed grafts covered significantly more surface area in FNRG animals with intermittent NTBC treatment compared to control animals (Fig. 2C, top; P < 0.01). Assuming that the grafts were spherical, we extrapolated graft volume on the basis of surface area measurements and calculated an average 11-fold graft expansion in NTBC-treated animals compared to control animals without liver injury (Fig. 2C, bottom; P < 0.05). To assay for active proliferation in tissue seeds 80 days after implantation, we double-stained sections using antibodies against both cytokeratin-18 and Ki67, a nuclear protein associated with cellular proliferation. We identified numerous cytokeratin-18 and Ki67 double-positive cells with round nuclei characteristic of hepatocytes (Fig. 2D, left), as well as rare double-positive cells actively undergoing mitosis (Fig. 2D, right). When compared with control animals, fourfold more cytokeratin-18 and Ki67 double-positive cells were observed in grafts from animals that received cycles of NTBC to induce liver injury and regeneration (Fig. 2D, graph; P < 0.01). To test whether our candidate tissue seeds also responded to regenerative cues after acute liver injury, we implanted seed grafts into the mesenteric fat of athymic mice. After a 1-week engraftment period, mice were subjected to two-thirds partial hepatectomy of the host liver to promote liver regeneration and given 5-ethynyl-2′-deoxyuridine (EdU) every 12 hours to label cells in the S phase of the cell cycle. One week after hepatectomy, animals were sacrificed, and engrafted tissues were excised, sectioned, and double-immunostained using antibodies that recognized EdU and cytokeratin-18 to identify hepatocytes in the S phase of the cell cycle. Grafts subjected to regenerative signals induced by hepatectomy injury contained more EdU and cytokeratin-18 double-positive cells compared to controls (fig. S3).

Hepatic function of ectopic liver seed grafts To evaluate the functional characteristics of the expanded hepatic seed grafts, we studied two major axes of liver function: synthesis and drug metabolism, and also performed a more global analysis of liver seed graft phenotypes. We first measured the protein synthesis capacity of the grafts by testing for human proteins in mouse serum. We detected human albumin in serum from engrafted mice treated with NTBC as early as day 3; the concentration of human albumin increased 50-fold from day 3 to the end point of the experiment in the NTBC-treated animals (Fig. 3, A and B). The maximum human albumin concentration detected in a single animal undergoing cycles of liver injury and regeneration was 105 μg/ml. Human serum albumin concentrations began to diverge between the treatment groups at about day 20, and liver seed grafts in animals with liver injury produced more albumin than those in control animals (Fig. 3, A and B; 10-fold difference at the end point of the experiment; P < 0.001). In addition to human albumin, blood drawn from NTBC-treated animals showed increased concentrations of human transferrin, α1-antitrypsin, and fibronectin relative to controls (Fig. 3C; P < 0.05). These results suggested that human hepatocytes in ectopic tissue seed grafts were functional and synthesized more human proteins when in the presence of regenerative signals induced by liver injury, compared to uninjured control animals. Fig. 3. Human liver tissue seed graft function. Mice were implanted with liver seed grafts, and the host mice were treated intermittently with NTBC to induce liver injury and repair (14 days off, followed by 3 to 4 days on; gray bars). (A) Blood samples were collected weekly by retro-orbital bleeding. The concentration of human albumin in mouse serum was greater in animals with liver injury compared to controls [P < 0.0001, one-way analysis of variance (ANOVA)]. (B) Human albumin concentration for each animal averaged across all time points. (C) The concentrations of human transferrin, α1-antitrypsin, and fibronectin in mouse serum were greater in animals receiving intermittent NTBC treatment (liver injury) compared to controls at 80 days after implantation into FNRG mice. (D) mRNA expression analysis of explanted liver seed grafts demonstrated that 47 of 50 liver-specific genes were expressed in explanted liver seed grafts (denoted “Seed graft”) compared to 18 of 50 human genes expressed in HUVEC and NHDF cell lines. (E and F) Genes from each of the major hepatic drug metabolism pathways were expressed in liver seed grafts at levels similar to those of the human liver. (G and H) Mice with liver seed grafts were injected with rifampin solution (25 mg/kg, intraperitoneally) or vehicle control daily for 3 days and, again, 1 hour before sacrifice. (I) Rifampin induced greater CYP3A4 expression in expanded liver seed grafts compared to mice injected with vehicle control. *P < 0.05, ***P < 0.001 (Student’s t test). To gauge the potential utility of liver seed grafts for studies of drug metabolism, we characterized the expression and induction of human drug-metabolizing enzymes and other key liver-specific proteins in expanded grafts implanted into FNRG mice. We first collected RNA for RNA sequencing (RNA-seq) analysis from liver seed grafts explanted from injured host mice, as well as from control samples comprising healthy human liver, primary human hepatocytes, HUVECs, and NHDFs. We then looked at the expression of 50 genes representing different hepatic gene classes that were expressed in both control human liver and primary human hepatocyte RNA samples. These gene groups included CYP3A4 and CYP2B6 for cytochrome P450 activity, SULT1A1/2A1 for sulfotransferase activity, SLCO1A2/1B1 for anion transporter activity, ABCB/ABCG for adenosine 5′-triphosphate (ATP)–binding transporters, APOB/APOE for lipoprotein biosynthesis, albumin for biosynthesis, and HNF4A/G encoding key transcription factors. Read counts across groups were normalized to human primary hepatocyte control RNA samples to create an expression heat map (Fig. 3D). A total of 47 of 50 of these liver-specific genes were expressed in explanted liver seed grafts, compared to 18 of 50 genes expressed in unexpanded HUVEC and NHDF RNA samples. Genes from each of the major hepatic drug metabolism pathways were expressed in liver seed grafts at similar levels to the human liver, including cytochrome P450 enzymes (Fig. 3E), sulfotransferases (Fig. 3F), and anion transporters and ATP-binding transporters (Fig. 3, G and H). In addition to hepatic gene expression studies, we tested the ability of liver seed grafts to up-regulate key drug metabolism enzymes in response to a known human CYP450 inducer (21). We administered rifampin or vehicle control to FNRG animals treated with NTBC bearing expanded liver seed grafts, euthanized the animals, and collected RNA from explanted liver seed grafts. We found that rifampin induced CYP3A4 expression in liver seed grafts, a highly liver-specific phenomenon indicative of mature hepatocyte function (Fig. 3I). Finally, we sought to interrogate the transcriptional profile of liver seed grafts more globally. We first used Ingenuity Pathway Analysis to assess the fraction of genes known to be downstream of given transcription factors that were differentially regulated between expanded liver seed grafts and HUVEC or NHDF control cells. This analysis identified distinct transcriptional regulation in liver seed grafts by hepatocyte transcription factors in the HNF1, HNF3, and HNF4 families, as well as C/EBP (CCAAT/enhancer-binding protein), compared to HUVEC or NHDF control cells (fig. S4A). This suggested that the hepatocytes present in expanded liver seed grafts displayed a lineage-appropriate phenotype. Furthermore, given that liver seed grafts were composed of primary hepatocytes, HUVECs, and NHDFs, we sought to test whether expression profiles from each of these three cell types were detectable in liver seed grafts after expansion. Hierarchical clustering of expression RNA-seq profiles obtained from samples of expanded liver seed grafts, pure human primary hepatocytes, human liver tissue, and pure populations of cultured NHDFs and HUVECs demonstrated that RNA expression in liver seed grafts clustered between those of primary hepatocytes/human liver samples and those of nonparenchymal HUVEC and NHDF cell lines. This was consistent with an intermediate phenotype driven by the presence of each of these three cell types within the expanded graft (fig. S4B).

Characterization of liver seed graft morphology after transplant Our earlier-generation engineered tissues, when implanted into uninjured mouse hosts, were characterized histologically by the presence of disperse hepatic aggregates within fibrin hydrogels upon explant (1, 2, 21). Here, we hypothesized that cells would self-organize in response to regenerative stimuli as the tissue seeds expanded. Immunohistological characterization revealed that the expanded liver seed grafts in NTBC-treated FNRG mice contained densely packed polyhedral cells resembling hepatocytes, many of which were binucleated (Fig. 4A; staining with hematoxylin and eosin). These cells stained positively for the hepatocyte markers cytokeratin-18 and arginase-1 (Fig. 4, B and C). Hepatocytes in expanded liver seed grafts were organized into dense aggregate-like units that, in some cases, exhibited structures reminiscent of hepatic cords in the normal human liver (Fig. 4B). Furthermore, hepatic units in expanded liver seed grafts were arranged within a syncytium of interconnected lacunae containing endovascular stroma and lined with collagen III, which lines hepatic cords in the space of Disse in the human liver (Fig. 4D, staining for reticulin). In addition, liver seed grafts were examined for the expression of multidrug resistance–associated protein 2 (MRP2; also known as ABCC2), which is selectively transported to the apical (that is, canalicular) domain of hepatocytes in the human liver. We observed that hepatocytes in expanded liver seed grafts exhibited polarized expression of MRP2 (Fig. 4C). Tissue seed grafts also contained bile canalicular–like structures between adjacent hepatocytes characteristic of normal liver structure (Fig. 4C), as well as larger vacuolar structures expressing MRP2 (Fig. 4C). Fig. 4. Characterization of human liver seed graft morphology. (A) Immunohistochemical staining of human liver tissue seed grafts from animals with liver injury sacrificed at day 80 revealed densely packed polyhedral cells resembling hepatocytes [stained with hematoxylin and eosin (H&E)] that were positive for both arginase-1 (B) and cytokeratin-18 (C). These human hepatocytes sometimes self-organized into cord-like structures (white star) (B). Hepatocytes were polarized, forming MRP2-positive bile canalicular–like structures between hepatocytes (white arrows, inset) and larger vacuoles lined with MRP2 (white stars) (C). (D) Hepatic units within the human liver seed grafts were surrounded by a syncytium of interconnected lacunae lined with collagen III as indicated by reticulin staining. (E) Human liver tissue seed graft sections stained with hematoxylin and eosin contained duct-like structures that resembled bile ducts (black arrows). (F) Cells in the ductal structures present in expanded liver seed grafts stained positive for cytokeratin-19, which is a marker for biliary epithelial cells but does not stain human hepatocytes (left). Cells organized in ductal structures stained positively for both cytokeratin-18 and cytokeratin-19 (white arrows, inset) (middle). Ductal structures were typically located adjacent to blood vessels lined with human CD31–positive endothelial cells and containing Ter-119–positive red blood cells (right). Scale bars, 25 μm. Further characterization with hematoxylin and eosin staining revealed that expanded tissue seed grafts also contained duct-like structures resembling bile ducts (Fig. 4E, arrows). To further examine whether biliary epithelial–like cells were present in ectopic grafted tissues, we immunohistochemically stained tissue sections for expression of both cytokeratin-18 (a cytokeratin expressed by hepatocytes and biliary epithelial cells) and cytokeratin-19 (a cytokeratin expressed by biliary epithelial cells but not hepatocytes). Cells organized in ductal structures stained positively for both cytokeratin-18 and cytokeratin-19, suggesting that these cells exhibited biliary epithelial–like characteristics (Fig. 4F). Notably, cytokeratin-18 and cytokeratin-19 double-positive ductal structures were typically located within connective tissue and adjacent to human CD31–positive blood vessels, many of which contained Ter-119–positive erythroid cells (Fig. 4F). To further confirm both the biliary epithelial cell–like phenotype and whether these cells were of human origin, we stained tissue sections for human cytokeratin-18 and a second cytokeratin expressed on biliary epithelial cells but not hepatocytes, human cytokeratin-7. Ungrafted mouse control liver tissue did not stain with either human marker, whereas positive control human liver tissue contained cytokeratin-18 and cytokeratin-7 double-positive cells in ductal structures (fig. S5). Ductal structures in ectopic liver seed grafts stained positive for both cytokeratin-18 and cytokeratin-7, further confirming that they were composed of human cells with an epithelial cell phenotype (fig. S5). We hypothesized that biliary epithelial–like cells identified in liver seed grafts may have arisen at least partially from contaminating biliary epithelial cells present in cryopreserved human hepatocyte samples. To test this hypothesis, we immunostained for cytokeratin-19 expression using cells from primary hepatocyte samples immediately upon thawing. We identified cytokeratin-19–positive cells in both of the human hepatocyte primary cell samples used in this study (0.16 and 0.13% of total cells in hu8085 and NON sample lots, respectively; fig. S6), suggesting that self-assembling biliary-like structures may have been at least partially derived from these cells. These results suggested that human biliary epithelial–like cells self-assembled to form ductal-like structures at an ectopic location within human liver tissue seeds and that these ductal structures were associated with other classic features of portal triads, such as vasculature and connective tissue.

Concomitant expansion of blood vessels lined with human endothelial cells Given that lacunae in the human liver form the vascular sinusoidal network that feeds hepatocytes with blood, we wondered whether interconnected lacunae observed in hepatic liver seed grafts contained red blood cells and whether regenerative cues would promote expansion of the total blood in the liver seed graft. We observed numerous cells resembling red blood cells in the lacunae of expanded seed grafts by hematoxylin and eosin staining (Fig. 4A). We further confirmed the identity of such cells by staining for Ter-119, an erythrocyte marker (Fig. 5A). We also quantified total blood area and observed that liver seed grafts from animals subjected to cycles of liver injury and regeneration contained significantly more red blood cells compared to those from control animals (Fig. 5A; P < 0.05), suggesting that the blood pool coordinately expanded with the expansion of hepatic seed grafts in animals with liver injury. Fig. 5. Vascularization of human liver seed grafts. (A) Lacunae between hepatocytes (red) contained Ter-119–positive red blood cells in liver seed grafts in animals with liver injury (left, green). Graph shows that liver seed grafts in animals with NTBC-induced liver injury and regeneration contained a greater number of red blood cells than did those in control animals. (B) Blood vessels containing Ter-119–positive red blood cells (white) and lined in part by human endothelial cells (red) were identified in tissue seed grafts (left). Liver seed grafts from animals with liver injury contained more human CD31 (huCD31)–positive blood vessels compared to grafts in control mice (right). Scale bars, 25 μm. *P < 0.05, ***P < 0.001 (Student’s t test). The presence of red blood cells in expanded tissue seed grafts suggested that vascular networks might be present in these grafts. Furthermore, several existing ectopic engraftment models have been shown to recruit host-derived vascular components, and human hepatocytes and vascular cells are known to expand in concert during liver regeneration (6, 28, 29). These observations led us to hypothesize that the prepatterned human endothelial cells within hepatic seed grafts may also expand in response to regenerative cues after liver injury. Thus, we immunostained expanded liver seed graft sections with antibodies recognizing human CD31 (endothelial cells), Ter-119, and arginase-1. Vessels lined in part by human endothelial cells were identified throughout the liver seed grafts that had expanded in FNRG mice treated with cycles of NTBC (Fig. 5B). Incubation of explanted graft sections in a solution containing lectins that bound specifically to human or mouse endothelium demonstrated that vessels were lined with both human and mouse endothelial cells (fig. S7). Many of these vessels contained Ter-119–positive erythroid cells (Fig. 5B). Blood vessels lined with CD31-positive human endothelial cells were located in the lacunae between and within hepatic units (Fig. 5B). Liver seed grafts in mouse hosts with liver injury showed more vessels containing human endothelial cells compared to liver seed grafts in control animals (Fig. 5B; P < 0.01). Ki67-positive human endothelial cells were present but were rare (less than one Ki67-positive and human CD31–positive cell per 1-mm section of liver seed graft). This suggested that most endothelial cells were not undergoing active cell cycle progression at the time of graft explant (fig. S8).