We wish to thank the Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, for providing the mice. We also thank S. Aoyama and Y. Adachi of the ADME (Absorption, Distribution, Metabolism, Excretion) &amp; Toxicology Research Institute, Sekisui Medical Company Ltd., Japan, and K. Kozakai and Y. Yamada for assistance with LC-MS/MS analysis. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan to Y-W.Z. (18591421, 20591531, and 23591872); by the Jiangsu innovative and entrepreneurial project for the introduction of high-level talent and the Jiangsu science and technology planning project (BE2015669); and by grants to H.T. for Strategic Promotion of Innovative Research and Development (S-innovation, 62890004) from the Japan Science and Technology Agency.

All animal experimental procedures were performed according to the Animal Protection Guidelines of Yokohama City University.
1. Generation of the Acute Liver Injury Mouse Model
<ol>
	<li>Add 1 ml of phenolized 0.85% NaCl solution (0.6 g phenol in 100 ml of 0.85% NaCl solution) to 1 mg diphtheria toxin (DT) to make a 1 mg/ml DT stock solution. Note: DT at a concentration of 1 mg/ml can be stored for approximately 2 years at 3 &#176;C to 8 &#176;C.</li>
	<li>Serially dilute the 1 mg/ml DT stock solution to 0.3 &#956;g/ml DT in phenolized 0.85% NaCl solution and prepare an aliquot of the 0.3 &#956;g/ml DT solution as a working solution. Note: This working solution should be freshly prepared.</li>
	<li>To conduct experiments using a sublethal dose (~ 50% lethality), administer 8-week-old mice a 1.5 &#956;g/kg dose of DT.
	<ol>
		<li>Perform an intraperitoneal injection of DT by holding the mouse in dorsal recumbency and insert the needle below the bend of the knees, left or right of the midline. Avoid the midline to prevent penetration of the bladder. Angle the needle at approximately 45&#176; to the body.</li>
		<li>Using an insulin syringe, inject the mice with 100 &#181;l of freshly prepared 0.3 &#956;g/ml DT per 20 g of mouse weight. For example, an 18-g mouse should receive 90 &#181;l of the 0.3 &#956;g/ml DT solution.</li>
	</ol>
	</li>
	<li>At 48 hr post DT injection, collect blood from the tail vein by placing the mouse in a restrainer and warming the mouse tail in a 37 &#176;C water bath for approximately 10 min to dilate the blood vessels. Then, make a 1-mm nick in the tail 2 cm from the tip with a sharp scalpel blade and collect the blood with a microcapillary tube.
	<ol>
		<li>Centrifuge the microcapillary tube containing the blood at 1,500 x g for 5 min and collect the serum by separating the supernatant and cell pellet.</li>
	</ol>
	</li>
	<li>Pipette 50 &#181;l of 1:20 diluted mouse serum onto GOT/AST-PIII slides. Measure the absorbance of the reaction product (blue dye) at 650 nm using a multi-purpose automatic dry-chemistry analyzer according to manufacturer&#39;s protocol. 
	NOTE: The read-out of aspartate aminotransferase (AST)/glutamic oxaloacetic transaminase (GOT) activity is displayed automatically. 2 days post DT injection, approximately 60% of the mice exhibited AST values between 12,000 - 16,000 IU/L and were considered to be suffering from acute liver damage. These mice were transferred to a new cage and used as cell transplant recipients.</li>
</ol>
2. Preparation of Human Hepatic Stem Cells
<ol>
	<li>Isolate human hepatic stem cells from human primary fetal liver cells with a cell sorter using the CDCP1, CD90, and CD66 cell antigens to obtain a CDCP1+CD90+CD66- subpopulation, then seed the isolated cell population on collagen IV-coated culture dishes, as previously reported15. Use human primary fetal liver cells of an embryonic age between weeks 14 and 18.</li>
	<li>Collect human hepatic stem cells cultured to 80% - 90% confluence on 100-mm culture dishes, aspirate the culture medium, wash the cells with 10 ml PBS, and then remove the PBS. Trypsinize the cells with 2 ml 0.05% trypsin/EDTA solution for 5 min at 37 &#176;C. Note: Cells at greater than 90% confluence are not suitable for cell transplantation since cell differentiation may influence their proliferative capability in mice.</li>
	<li>Monitor the status of cell dispersion under a microscope. When the cells appear to be floating or flowing freely, stop the enzymatic digestion by adding 8 ml of DMEM/F12 containing 10% FBS.</li>
	<li>Suspend the cells slowly using a 10-ml serological pipette and transfer the cells into a 15-mL conical tube. Centrifuge the cells for 5 min at 100 x g and 4 &#176;C.</li>
	<li>Carefully resuspend the cell pellet in 10 ml DMEM/F12 containing 10% FBS and determine cell numbers using a microscope counting chamber.</li>
	<li>Divide the cells into 1.0 x 106 cells per 50 &#181;l of PBS aliquots for each individual mouse and store on ice until transplantation.</li>
</ol>
3. Intrasplenic Transplantation of Human Hepatic Stem Cells
<ol>
	<li>Place a clean cage on a 37 &#176;C electric heating pad.</li>
	<li>Anesthetize the mouse using isoflurane inhalation (1 - 1.5% (vol/vol) in 2 L of oxygen per min) by placing it underneath the nozzle. Alternatively, use tubes containing gauze soaked with isoflurane.
	<ol>
		<li>Ensure that the mouse is anesthetized by lightly pinching the rear footpad. If a knee jerk is elicited, place the mouse back in the chamber. Use vet ointment on the eyes to prevent dryness while under anesthesia.</li>
	</ol>
	</li>
	<li>Shave the surgical site using electric clippers and apply 70% (vol/vol) ethanol and povidone-iodine to sterilize. Then make a 1-cm skin incision in the left flank just below the costal border of the ribs, followed by an incision on the abdominal wall, as well as the peritoneum.</li>
	<li>Carefully expose the spleen. Use a 100-&#956;l microinjection syringe with a 32-G 1/2-inch needle to directly inject 1.0 x 106 human hepatic stem cells in 50 &#181;l PBS into the spleen of each mouse. Tilt the needle at a 5&#176; angle for injection.
	<ol>
		<li>Ensure that the depth of injection is less than half the thickness of the spleen. The needle should enter the spleen at one end (the head) and deposit the cells at the other end (the tail).</li>
	</ol>
	</li>
	<li>To prevent leakage of the cells following injection, gently apply pressure using a finger for 2 min and then remove the needle.</li>
	<li>Place the spleen back into the mouse body and close the cavity with a normal running suture for the musculature and skin.</li>
	<li>Transfer the mice into a 37 &#176;C pre-warmed cage immediately following transplantation. To ensure the mouse is able to breathe comfortably, place the mouse on its side in the cage, avoiding contact between the cage bedding and the site of surgery.</li>
	<li>Monitor the mice until the anesthesia wears off to ensure the sutures remain closed and the mice return to pre-surgery conditions.</li>
	<li>Ensure the mice are provided with normal drinking water and food. After 1 hr, return the cage to the mouse room of the animal center and monitor the mice daily.</li>
</ol>
4. Detection of Transplanted Human Hepatic Stem Cell-Derived Hepatocytes in the Mouse Liver
Note: For the following procedures, euthanize all animals using an overdose of ketamine and xylazine followed by cervical dislocation.
<ol>
	<li>At 4 - 6 weeks post transplantation, euthanize the mice and open the abdominal cavity by simultaneously cutting the cutis and fascia using surgical scissors.</li>
	<li>Dissect the connective tissue above the peritoneum, using the scissors as a spreader, and cut the peritoneum along the linea alba to open the peritoneal cavity.</li>
	<li>Lift the sternum with forceps, puncture the diaphragm, and cut through each side of the sternum up through the cervical girdle.</li>
	<li>Sever the vena cava on the thoracic side of the liver and pull the esophagus through the liver in the anterior direction. Remove the diaphragm in order to remove the liver. 
	Note: Be careful to keep the tips of the scissors pointed upward in order to prevent any damage to the thoracic organs underneath. The thymus has a tendency to occasionally cling to the dorsal side of the sternum and this must be carefully avoided.</li>
	<li>Using the diaphragm as a handle, start pulling the liver out of the abdominal cavity. The interior vena cava will be holding the liver in place. Cut the interior vena cava; be careful not to prematurely liberate the right adrenal. 
	Note: Mice have a four-lobed liver consisting of a median lobe, left lobe, right lobe, and the caudate lobe. The gall bladder is suspended in the small bifurcation of the median lobe.</li>
	<li>Separate the lobes from each other at the junctions and embed them in optimal cutting temperature (OCT) compound according to the manufacturer&#39;s protocol. Freeze the OCT containing the liver tissue in the metal grids on the cryostat.</li>
	<li>Cut 5-&#181;m-thick sample sections using a cryostat at -18 &#176;C and mount them on RT microscope slides. Store the stock at -80 &#176;C. Prepare the slides for hematoxylin and eosin (H&#38;E) and immunofluorescence staining according to previously reported procedures16.</li>
</ol>
5. Detection of Human Albumin Secretion and Calculation of the Chimeric Rate
<ol>
	<li>To measure human liver reconstitution, perform human albumin ELISA using serum and a human albumin ELISA kit according to the manufacturer&#39;s instructions.</li>
	<li>Calculate the humanized liver chimeric rate by real-time PCR analysis of expression levels in the whole-humanized liver. Determine the relative expression level ratio of human-specific actin to human-mouse cross actin (ratio 1) and the ratio of mouse-specific actin to human-mouse cross actin (ratio 2). The chimeric rate is calculated as ratio 1/(ratio 1 + ratio 2).</li>
</ol>

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Ther. 344, 388-396 (2013).</li><li>Suemizu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+humanized+model+of+liver+using+NOD/Shi-scid+IL2Rgnull+mice.">Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice.</a> Biochem. Biophys. Res. Commun. 377, 248-252 (2008).</li><li>Tateno, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+completely+humanized+liver+in+mice+shows+human-type+metabolic+responses+to+drugs.">Near completely humanized liver in mice shows human-type metabolic responses to drugs.</a> Am. J. Pathol. 165, 901-912 (2004).</li><li>Hasegawa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reconstituted+'humanized+liver'in+TK-NOG+mice+is+mature+and+functional.">The reconstituted 'humanized liver'in TK-NOG mice is mature and functional.</a> Biochem. Biophys. Res. Commun. 405, 405-410 (2011).</li><li>Azuma, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+expansion+of+human+hepatocytes+in+Fah&#8722;/&#8722;/Rag2&#8722;/&#8722;/Il2rg&#8722;/&#8722;+mice.">Robust expansion of human hepatocytes in Fah&#8722;/&#8722;/Rag2&#8722;/&#8722;/Il2rg&#8722;/&#8722; mice.</a> Nat. Biotechnol. 25, 903-910 (2007).</li><li>Rhim, J. A., Sandgren, E. P., Degen, J. L., Palmiter, R. D., Brinster, R. 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The authors have no competing financial interests to disclose.

Recent studies have shown that the mouse liver can be repopulated with human hepatocytes, including adult hepatocytes and proliferative hepatic stem cells17. These repopulated livers have been used as preclinical experimental models for drug metabolism testing and drug discovery and development18; in addition, they have provided an in vivo environment for cell maturation and differentiation19. The major aim of the present study was to generate a novel liver disease mouse model that allowed human immature hepatocyte proliferation, maturation, and differentiation, for the acquisition of drug metabolism activities.
The course of intrasplenic transplantation has been studied extensively. Cells transplanted into the spleen of rats and mice survive throughout their average life span. The intrasplenic transplantation protocol has proven to be very useful in investigating specific research questions; many laboratories worldwide have attempted to utilize this protocol as a highly reproducible and reliable model for hepatic cell transplantation because of the improved mice survival and liver regeneration. However, implanting cells into the splenic pulp poses a risk, since the majority of implanted cells will translocate to the liver where they can occlude portal branches. Even though this occlusion can be temporary and might not affect a healthy liver environment, in cases of preexisting liver damage, this procedure could further reduce liver function and increase portal hypertension and might also negatively influence cell engraftment. Although we have successfully applied hepatocyte transplantation using intrasplenic transplantation, several issues warrant further study, especially the subject of potential new cell sources. Moreover, when performing intrasplenic hepatocyte transplantation, the cell loading limit must be strictly followed in order to prevent potential disturbance by remnant hepatic microcirculation. The DT receptor has been identified as a membrane-anchored form of the heparin-binding EGF-like growth factor (HB-EGF precursor)20. Whereas HB-EGF precursors derived from toxin-sensitive animals, such as humans and monkeys, bind DT and function as toxin receptors, HB-EGF precursors from mice and rats do not bind DT21. The transgenic Alb-TRECK/SCID mouse model expresses human HB-EGF-like receptors under the control of a liver cell-specific albumin promoter; following the administration of DT, these mice develop fulminant hepatic failure due to conditional ablation of hepatocytes, providing space for donor cell residency and proliferation. Human hepatocytes, even at an immature phase, are known to possess extensive proliferative capacity in vitro, although they lose their drug metabolism functions, limiting their preclinical applications22. Histological and immunohistochemical findings obtained following the transplantation of human HpSCs into Alb-TRECK/SCID mice with lethal fulminant hepatic failure indicated that the human HpSCs could expand and reconstitute the damaged mouse liver structures; the repopulation rate in some mice was nearly 100%.
In our experiments, human HpSC transplantation promoted mouse survival and resulted in increased secretion of human ALB into mouse sera16. These cells also matured and differentiated in vivo and exhibited human drug metabolism activities, indicating that human HpSC-derived humanized mice livers are similar to mature and functional &#34;human organs&#34; and have potential applications in drug development. These results further confirm that the Alb-TRECK/SCID mouse is an ideal model for humanized liver generation. Despite the prospects and advantages of Alb-TRECK/SCID mice as an ideal model for human hepatic stem cell-derived humanized liver generation, they possess one unique disadvantage; when human adult hepatocytes were transplanted into three Alb-TRECK/SCID mice treated with a single dose of DT, no humanized livers were generated, possibly because human adult hepatocytes lack proliferative capacity. Additional doses of DT cannot be administered post transplantation of human adult hepatocyte; since the DT receptor (HB-EGF) in Alb-TRECK/SCID mice hepatocytes is under the control of the albumin promoter, additional DT treatment will destroy the transplanted human adult hepatocytes in the mouse liver.
To the best of our knowledge, this is the first model to generate a humanized liver from immature human cells. Cell replacement rate post-transplant is influenced by the recipient micro-environment (e.g., hepatic niche) and donor cell properties, such as mature vs. immature and proliferative and differential capability. Combining Alb-TRECK/SCID with stem cell transplantation will provide optimized conditions and advantages for the generation of humanized livers. This also constitutes a powerful tool for transplantation of fetal liver cells and potentially as well as iPS or ES-derived hepatic cells since prior to DT treatment, the Alb-TRECK/SCID animals are maintained in a healthy state, liver damage is inducible at any point, and the mice breed normally without renal damage. In contrast, uPA/SCID mice exhibit high neonatal mortality and are difficult to breed. Furthermore, overexpression of urokinase in the animals can lead to severe bleeding and renal damage and thus there is a limited window of opportunity for cell transplantation prior to weaning; Alb-TRECK/SCID can be used for cell transplantation at any age.
The successful generation of a humanized liver mouse requires the following critical steps: 1) Liver injury should be limited to sub-lethal levels (usually by using the lethal dose, 50%); 2) The human primary fetal liver cells should be maintained in a sub-confluent status no greater than 90%; 3) Cell leakage post intrasplenic-transplantation should be prevented; and 4) The levels of human albumin in mouse sera should be monitored at regular intervals. A comprehensive understanding of the critical factors involved in the process of humanized mouse liver generation might contribute to further modifications or new developments utilizing rats or other animals. Possible adaptations include: the induction of kidney injury for examining development and repair with human nephrons, their progenitors, and collecting duct cells and the self-assembly of human nephrons into three dimensional structures in the mouse body; studying the repair of islet damage in the pancreas using pancreatic stem/progenitor cells derived from directly reprogrammed, iPS, or ES cells. Examples of process modifications include: controlling tissue or organ injury with toxins or chemicals such as retrorsine, streptozotocin, or cisplatin to facilitate the competition of donor cells with resident cells; avoiding differential or decreasing proliferation in order to enhance the proliferative capability of the donor cells, thus promoting the replacement of recipient cells, such as ES or iPS- derived cells; selecting a suitable transplant sites and controlling cell infusion to ensure efficient transplantation, for example, using the portal or tail vein for entopic transplantation and the mesentery and renal capsule for ectopic transplantation; monitoring replacement status via the expression of tissue or organ secreted proteins representative of stem/progenitor or immature cells transplant and cancer cell tracing, such as alpha fetoprotein or transferrin.
In summary, Alb-TRECK/SCID mice constitute a suitable model for human hepatic stem cell transplantation, as they provide a beneficial environment that allows for differentiation into mature hepatocytes. This novel model has tremendous applicative potential, not only for ex vivo expansion of human hepatocytes but also for drug screens and testing candidate therapeutic drugs for liver toxicity and metabolism. In addition, these results will aid in the advancement of future studies examining the clinical application of this technique to human hepatic cell transplantation.

Mice are commonly used for pharmaceutical testing since biomedical research in humans is restricted1; however, these models are not always useful since they may inaccurately simulate the effects observed in humans. Most drugs in current medical use are metabolized primarily in the liver. However, the same drug can be metabolized into different metabolites in mouse and human livers because of inter-species differences. Thus, it is often difficult to determine during development whether a potential drug poses any risks for clinical applications2,3. 
To address this problem, "humanized" mouse livers have been developed by growing human liver cells inside mice4-6; these models exhibit drug responses similar to those observed in the human liver. The primary mouse models currently used for humanized liver generation include uroplasminogen activator (uPA+/+) mice4,7, fumarylacetoacetate hydrolase (Fah&#8722;/&#8722;) mice6, and the recently reported thymidine kinase (TK-NOG) mice. 
However, previous reports have shown that transplanted human immature cells or stem cells are less competitive than adult human hepatocytes in Alb-uPA tg(+/&#8722;)Rag2(&#8722;/&#8722;) mouse livers8-10. Moreover, Fah&#8722;/&#8722; mice provide a growth advantage only for differentiated hepatocytes and not for immature liver progenitor cells11. The transplantation of human hepatic stem cells (HpSCs) into TK-NOG mice in the lab has been unsuccessful. Hence, no useful mouse model for the efficient engraftment of human immature liver cells currently exists. 
Thus, we developed a novel Alb-TRECK/SCID mouse model that could be efficiently repopulated with human immature hepatocytes. This transgenic mouse model expresses human heparin-binding EGF-like growth factor (HB-EGF) receptors under the control of a liver cell-specific albumin promoter. Following the administration of diphtheria toxin (DT), these mice develop fulminant hepatitis due to conditional ablation of hepatocytes, enabling donor cell residency and proliferation12. Although mouse hepatocytes have been successfully transplanted into Alb-TRECK/SCID mice in previous studies13,14, the generation of a humanized liver using Alb-TRECK/SCID mice has yet to be reported. 
In this study, humanized livers were generated in Alb-TRECK/SCID mice via transplantation of HpSCs. This humanized liver provides an in vivo environment for universal stem cell differentiation and the ability to predict human drug metabolism patterns and drug-drug interactions.

<table><tbody><tr><td>Human albumin</td><td>Sigma</td><td>A6684</td><td>Mouse</td></tr><tr><td>Human CK19</td><td>Dako</td><td>M088801</td><td>Mouse</td></tr><tr><td>Human nuclei</td><td>Millipore</td><td>MAB1281</td><td>Mouse</td></tr><tr><td>Human CK8/18</td><td>Progen</td><td>GP11</td><td>Guinea pig</td></tr><tr><td>CDCP1</td><td>Biolegend</td><td>324006</td><td>Mouse</td></tr><tr><td>CD90</td><td>BD</td><td>559869</td><td>Mouse</td></tr><tr><td>CD66</td><td>BD</td><td>551479</td><td>Mouse</td></tr><tr><td>GOT/AST-PIII</td><td>Fujifilm</td><td>14A2X10004000009</td><td></td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>11320-033</td><td></td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520</td><td></td></tr><tr><td>0.05% Trypsin-EDTA&amp;nbsp;</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Diphtheria Toxin</td><td>Sigma</td><td>D0564-1MG</td><td></td></tr><tr><td>Human Albumin ELISA Kit</td><td>Bethyl Laboratories</td><td>E88-129</td><td></td></tr><tr><td>Syringe (1 ml)</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>32G 1/2&quot; needle</td><td>TSK</td><td>PRE-32013</td><td></td></tr><tr><td>O.C.T.Compound(118 ml)</td><td>Sakura Finetek Japan</td><td>4583</td><td></td></tr><tr><td>MoFlo high-speed cell sorter</td><td>Beckman Coulter</td><td>B25982</td><td></td></tr><tr><td>DRI-CHEM 7000</td><td>Fujifilm</td><td>14B2X10002000046</td><td></td></tr></tbody></table>

generation of a humanized mouse liver using human hepatic stem cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Alb-TRECK/SCID mice hepatocytes express the human DT receptor HB-EGF gene under the control of an albumin promoter and exhibit cytotoxic effects following DT administration12. To evaluate the effects of DT treatment on liver injury, DT doses of 1.5 &#956;g/kg were injected into 8-week-old Alb-TRECK/SCID mice and the pathological changes in the liver 48 hr post DT administration were histologically assessed. Compared with control mice (not treated with DT), the DT-treated mice exhibited a disorganized hepatic architecture showing a correction for congestion with increased serum AST activity. In addition, the hepatocytes of DT-treated mice had multiple, deeply stained acidophilic cytoplasmic inclusions together with dark nuclei (most probably apoptotic hepatocytes); some other hepatocytes appeared preserved or exhibited a vacuolated cytoplasm with dark nuclei with little or no portal vein inflammation (Figure 1). These findings are concordant with those of a previous study that showed that Alb-TRECK/SCID mice represent an ideal lethal fulminant hepatic failure model and can be generated using only a single DT injection12.
Human HpSCs can be used for long-term in vitro culturing and exhibit uniform cell morphology with some ALB and fewer CK19-positive cells. At 6 weeks post transplantation into the mouse livers, the human HpSCs were subjected to basic examination under a microscope and evaluated histologically; human HpSCs were observed to have reconstituted the liver structure by replacing the original mouse hepatocytes (Figure 2). Macroscopically, at 4 days post human HpSC transplantation, small human hepatic clusters that were uniformly distributed around the liver were detected; at 45 days post transplantation, these had proliferated into large clusters (Figure 3). Whole mouse livers reconstituted with human hepatocytes were designated &#34;humanized livers.&#34; These results indicate that mice with lethal fulminant hepatic failure that undergo human HpSC transplantation may exhibit liver structures similar to those observed in normal mouse livers.
Prior to testing whether the immature human hepatocytes transplanted into mice had the potential for differentiation and could function in vivo, the presence of human hepatocytes in mouse livers was first confirmed by immunohistochemical analysis at approximately 6 weeks post human HpSC transplantation. Liver sections from mice with 60% human cell repopulation that specifically and positively co-stained with human nuclei and human cytokeratin 8/18 (CK8/18) antibodies were found to contain donor cell-derived human hepatocytes, whereas the original control mice livers were negative for these markers (Figure 4).
To assess the degree of cell differentiation in vivo, the expression of human albumin (ALB) and human cytokeratin 19 (CK19) was assessed immunohistochemically. Human HpSC-derived human hepatocytes were well differentiated in the mouse livers and exhibited upregulated human ALB expression. Human ALB-positive and CK19-negative hepatocytes resembled functional hepatocytes, whereas cells that positively co-stained with human ALB and CK19 exhibited bipotential capability for differentiating into hepatocytes and cholangiocytes (Figure 5). A large-scale scanning method used to analyze all human HpSC-derived humanized liver lobes revealed the presence of multiple round and colony-like clusters surrounding the liver lobes with clear human nuclei and CK8/18 expression (Figure 6), demonstrating the colony-forming capability of human HpSCs in the humanized livers. The repopulation level reached up to 90% one month following transplantation (Figure 7A) and human albumin secretion was detected in the repopulated mice livers (Figure 7B), indicating that human HpSCs could successfully reconstitute the Alb-TRECK/SCID mouse liver. These results demonstrate that human HpSCs have high potential for differentiation into functional hepatocytes in vivo in response to the rescue of defective mouse liver functions.
<img alt="Figure 1" src="/files/ftp_upload/54167/54167fig1.jpg" /> 
Figure 1. Liver Damage Occurs in Alb-TRECK/SCID (Toxin Receptor Mediated Cell Knockout) Mice Following Diphtheria Toxin (DT) Injection. Histology (hematoxylin-eosin staining) of mouse livers without and with DT administration after 48 hr. DT dose: 1.5 &#956;g/kg, serum AST activity of normal liver (left panel): 30 IU/L; serum AST activity of DT-treated liver (right panel): 15000 IU/L. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54167/54167fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54167/54167fig2.jpg" /> 
Figure 2. Transplantation of Human Hepatic Stem Cells into Alb-TRECK/SCID Mice with Fulminant Hepatic Failure. Macroscopic view (left panel) and histological analysis (hematoxylin-eosin staining, right panel) of humanized livers with human HpSCs at 6 weeks post transplantation. m, mouse liver region; h, human donor cell-derived human region. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54167/54167fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54167/54167fig3.jpg" /> 
Figure 3. Macroscopic Analysis of Humanized Livers Derived from Human Hepatic Stem Cells Labeled with Green Fluorescent Protein. High-magnification images of the liver structure 4 days (left panel) and 45 days (right panel) following transplantation with human hepatic stem cells (HpSCs). Human hepatic stem cells (HpSCs) were labeled with enhanced green fluorescent protein (EGFP) using a lentivirus vector. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54167/54167fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54167/54167fig4.jpg" /> 
Figure 4. Characterization of Human Hepatic Stem Cell-Derived Human Hepatocytes in Alb-TRECK/SCID Mice. Immunohistochemical analysis distinguishing between human hepatocytes stained with anti-human CK8/18 (green) antigen and anti-human nuclear antigen (aqua blue) in human hepatic stem cell-derived humanized livers at 6 weeks post transplantation. m, mouse liver region; h, human donor cell-derived human liver region. White dashed line: mouse liver region vs. human liver region. Nuclei were counterstained with 4&#8242;,6-diamidino-2-phenylindole (blue). Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54167/54167fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54167/54167fig5.jpg" /> 
Figure 5. Human Hepatic Stem Cells Differentiation in Humanized Livers of Alb-TRECK/SCID Mice. Immunohistochemical analyses of human albumin and human CK19 in HpSC-derived livers at 6 weeks post transplantation. Nuclei were counterstained with 4&#8242;,6-diamidino-2-phenylindole (blue). Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54167/54167fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54167/54167fig6.jpg" /> 
Figure 6. Multiple Clusters of Human Hepatic Stem Cell-Derived Cells are Present in Alb-TRECK/SCID Mice with Humanized Livers. Multiple large clusters derived from human hepatic stem cells were identified in the liver lobes at 6 weeks post transplantation by immunohistochemical analysis for human CK8/18 (green) and human nuclei (aqua blue). Scale bars = 1,000 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54167/54167fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54167/54167fig7.jpg" /> 
Figure 7. Humanized Liver Repopulation Rate and Human Albumin Secretion in Alb-TRECK/SCID Mice. (A) Chimeric rate in Alb-TRECK/SCID mice 1 month following human hepatic stem cell transplantation; data is presented as mean &#177; SEM (n = 12). Mann-Whitney test: P &#60; 0.0005 for Sham and human HpSCs. (B) Detection of human albumin in mouse sera one month following donor cell transplantation. Data = mean &#177; SEM (n = 5). Mann-Whitney test: P = 0.0313 for Sham and human HpSCs. ND: undetectable; Sham: mice transplanted with PBS; HpSCTx: mice transplanted with human HpSCs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54167/54167fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells at JoVE.com

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

generation-of-a-humanized-mouse-liver-using-human-hepatic-stem-cells

Nanyang Technological University

Research

JoVE Journal

Medicine

10.8K Views.  Yokohama City University. Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

Video: Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Immunology and Infection

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Cancer Research

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Developmental Biology

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Biology

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Bioengineering

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model

Source: Daharsh, L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59773">A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System.</a>&#160;J. Vis. Exp. (2019)
This video demonstrates the method for generating a double humanized bone BLT (marrow, liver, and thymus) mouse with a functional human immune system and a stable engrafted human-like gut microbiome.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mice ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

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