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 stoc...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatic+stem+cell,+method+for+preparation+of+the+same,+method+for+induction+of+differentiation+of+the+same,+and+method+for+utilization+of+the+same.">Human hepatic stem cell, method for preparation of the same, method for induction of differentiation of the same, and method for utilization of the same.</a> Japan Patent. , (2015).</li><li>Zhang, R. -. R., 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=Human+hepatic+stem+cells+transplanted+into+a+fulminant+hepatic+failure+Alb-TRECK/SCID+mouse+model+exhibit+liver+reconstitution+and+drug+metabolism+capabilities.">Human hepatic stem cells transplanted into a fulminant hepatic failure Alb-TRECK/SCID mouse model exhibit liver reconstitution and drug metabolism capabilities.</a> Stem Cell Res. 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B. 2, 549-561 (2012).</li><li>Nowak, G., 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=Identification+of+expandable+human+hepatic+progenitors+which+differentiate+into+mature+hepatic+cells+in+vivo.">Identification of expandable human hepatic progenitors which differentiate into mature hepatic cells in vivo.</a> Gut. 54, 972-979 (2005).</li><li>Naglich, J. G., Metherall, J. E., Russell, D. W., Eidels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+a+diphtheria+toxin+receptor:+identity+with+a+heparin-binding+EGF-like+growth+factor+precursor.">Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.</a> Cell. 69, 1051-1061 (1992).</li><li>Mitamura, T., Higashiyama, S., Taniguchi, N., Klagsbrun, M., Mekada, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diphtheria+toxin+binds+to+the+epidermal+growth+factor+(EGF)-like+domain+of+human+heparin-binding+EGF-like+growth+factor/diphtheria+toxin+receptor+and+inhibits+specifically+its+mitogenic+activity.">Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity.</a> J. 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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 th...

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 border="0" cellpadding="0" cellspacing="0" width="538">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Human albumin</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:124px;">A6684</td>
			<td style="width:60px;">Mouse</td>
			<td style="width:60px;">IgG2a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human CK19</td>
			<td>Dako</td>
			<td>M088801</td>
			<td>Mouse</td>
			<td>IgG1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human nuclei</td>
			<td>Millipore</td>
			<td>MAB1281</td>
			<td>Mouse</td>
			<td>IgG1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human CK8/18</td>
			<td>Progen</td>
			<td>GP11</td>
			<td>Guinea pig</td>
			<td>Polyclonal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CDCP1</td>
			<td>Biolegend</td>
			<td>324006</td>
			<td>Mouse</td>
			<td>IgG2b</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD90</td>
			<td>BD</td>
			<td>559869</td>
			<td>Mouse</td>
			<td>IgG1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD66</td>
			<td>BD</td>
			<td>551479</td>
			<td>Mouse</td>
			<td>IgG2a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GOT/AST-PIII</td>
			<td>Fujifilm</td>
			<td>14A2X10004000009</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Biowest</td>
			<td>S1520</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.05% Trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diphtheria Toxin</td>
			<td>Sigma</td>
			<td>D0564-1MG</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Albumin ELISA Kit</td>
			<td>Bethyl Laboratories</td>
			<td>E88-129</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe (1ml)</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">32G 1/2&#34; needle</td>
			<td>TSK</td>
			<td>PRE-32013</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T.Compound(118ml)</td>
			<td>Sakura Finetek Japan</td>
			<td>4583</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MoFlo high-speed cell sorter</td>
			<td>Beckman Coulter</td>
			<td>B25982</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DRI-CHEM 7000</td>
			<td>Fujifilm</td>
			<td>14B2X10002000046</td>
			<td></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 e...

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 ...

The overall goal of this surgical stem cell transplant procedure is to generate a novel chimeric mouse model with human liver function using human immature hepatic stem progenitor cells that allow cell proliferation, maturation and differentiation for the acquisition of drug metabolism. This method provides a powerful tool as to create a micro-environment to support human donor hepatic stem cell progenesis to expand, differentiate and reconstitute the injured mouse recepient liver. The main advantage of this technique is that it matured proliferated cells can be used and that the liver almost completely repopulates.To begin, make a one milligram per milliliter diphtheria toxin or DT stock solution by adding one milliliter of Phenolized 0.85%sodium chloride solution to one milligram of DT.Perform an intraperitoneal injection of DT by holding an albtrek scid mouse in dorsal recumbency and below the bend of the knees left or right of the midline, insert the needle at a 45 degree angle to the body. Using an insulin syringe, inject 100 microliters of freshly prepared 0.3 microgram per milliliter of DT per 20 gram mouse. At 48 hours post DT injection, place the mouse in a restrainer and warm the tale in a 30 degree Celsius water bath for approximately 10 minutes to dilate the blood vessels.Then two centimeters from the tip of the tale, use a sharp scalpel blade to make a one millimeter nick in the tale and use a capillary tube to collect the blood. Centrifuge the microcapillary tube containing the blood at 1500 times G for five minutes and collect the serum by separating the supernatant from the cell pellet. Pipette 50 microliters of a one to 20 dilution of the serum onto GOT ASTP3 slides.Then using a multi-purpose automatic dry chemistry analyzer according to the manufacturer's protocol, measure the absorbance of the reaction product at 650 nanometers. Using a cell sorter, isolate human hepatic stem cells from human primary fetal liver cells with the CDCP1, CD90 and CD66 cell antigens to obtain a CDCP1 positive, CD90 positive, CD66 negative subpopulation. Seed the cells onto collagen four coded culture dishes and incubate at 37 degrees Celsius.When the cells reach 80 to 90%confluence, use PBS to wash the cells once. Then add 0.05%Trypsin EDTA and incubate at room temperature. When the cells appear to be floating or flowing freely under a microscope stop the enzymatic digestion by adding eight milliliters of DMF12 containing 10%FBS.With a 10 milliliter serological pipette, slowly suspend the cells before transferring them to a 15 milliliter conical tube. Centrifuge the cells at 100 times G and four degrees Celsius for five minutes. Using 10 milliliters of DMF12 with 10%FBS, carefully resuspend the cell pellet and use a cell count instrument to determine the cell numbers.Divide the cells into aliquots of one times 10 to the sixth cells per 50 microliters of PBS for each mouse and store on ice until transplantation. After anesthetizing a mouse according to the text protocol, check for a reflex by lightly pinching the rear foot pad. Then apply vet ointment to the eyes to prevent dryness.With electric clippers, shave the surgical site and apply 70%ethanol and povidone-iodine to disinfect. Make a one centimeter skin incision in the left flank just below the costal border of the ribs. Then make an incision on the left abdominal wall followed by an incision on the peritoneum.Next, carefully expose the spleen. Then at a five degree angle, insert a 100 microliter micro-injection syringe with a 32 gauge one half inch needle at a depth half the thickness of the spleen and from one end of the organ to the other directly inject one times 10 to the sixth human hepatic stem cells in 50 microliters of PBS in the organ and remove the needle. To prevent leakage of the cells following injection, use a finger to gently apply pressure for two minutes.Place the spleen back into the mouse body and use a normal running suture for the musculature and skin to close the cavity. Immediately transfer the mouse on its side into a 37 degree Celsius pre-warmed cage. Avoid contact between the cage bedding and site of surgery and monitor the animal according to the text protocol.At four to six weeks post transplantation, after euthanizing the mouse according to the text protocol, use surgical scissors to open the abdominal cavity by simultaneously cutting the cuteous and fascia. Using the scissors as a spreader, dissect the connective tissue above the peritoneum along the linea alba to open the peritoneal cavity. With forceps, lift the sternum.Then puncture the diaphragm and cut through each side of the sternum up through the cervical girdle. Severe the vena cava on the thoracic side of the liver and pull the esophagus through the liver in the anterior direction. Remove the diaphragm and then using it as a handle, start pulling the liver out of the abdominal cavity.The inferior vena cava will be holding the liver in place. Therefore, cut the inferior vena cava taking care not to prematurely liberate the right adrenal. Separate the lobes of the liver from each other at the junctions and embed them in OCT according to the manufacturer's protocol.Freeze the OCT containing the liver tissue in the metal grids on the cryostat. With the cryostat, cut five micrometer thick sample sections at minus 18 degrees Celsius and mount them on room temperature microscope slides. Store the stock at minus 80 degrees Celsius.Finally, prepare the slides for H&E staining according to previously reported procedures and ELISA according to the text protocol. As shown in these H&E stained sections, DT treated mice exhibited a disorganized hepatic architecture and displayed a correction for congestion with increased serum AST activity. At six weeks post transplantation, human hepatic stem cell derived cells reconstituted the liver structure by replacing the residential mouse hepatocytes.Macroscopically, at four days post-transplantation, small human hepatic clusters that were uniformly distributed around the liver were detected. At 45 days, the clusters had proliferated into large clusters. As seen here, the presence of human hepatocytes in mouse livers was first confirmed at six weeks post-transplantation by staining with human nuclei and cytokeratin 8/18 antibodies.Human ALB positive and CK19 negative hepatocytes resembled functional hepatocytes whereas cells that positively co-stained for human ALB and CK19 exhibited bi-potential capability for differentiating into hepatocytes and cholangiocytes. The presence of multiple round clusters surrounding the liver lobes with clear human nuclei and CK8/18 expression was detected demonstrating the colony forming capability of human hepatic stem cells in the humanized livers. Finally, the repopulation level reached up to 90%one month following transplantation and human Albumin secretion was detected in the repopulated mouse livers.Once mastered, surgical cell transplantation can be done in 10 minutes if it is performed properly. While attempting this procedure, it's important to remember that liver injure should be restricted to supple lizer liver. Under cell leakage, cell transplantation should be prevented.Combined with this procedure, other methods like gene transfection for inviro direct reprogramming with human cells can be performed in order to explore human cell convergence with our current model. After its development, this technique paved the way for researchers in the hepatology field to explore human drug metabolism, drug drug interaction, hepatic toxicity, and hepatisis virus in a humanized liver model. After watching this video, you should have a good understanding of how to isolate and expand the hepatic stem cell, induce liver injury, and transplant a cell via the spleen to generate a chimeric mouse model with human liver function.Don't forget that diphtheria toxin can be controlled and important and precautions such as avoiding necrosis of the heart and the liver should always be taken while performing this procedure.

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

Research

JoVE Journal

Medicine

10.8K 조회수.  Yokohama City University. The overall goal of this surgical stem cell transplant procedure is to generate a novel chimeric mouse model with human liver function using human immature hepatic stem progenitor cells that allow cell proliferation, maturation and differentiation for the acquisition of drug metabolism. This method provides a powerful tool as to create a micro-environment to support human donor hepatic stem cell progenesis to expand, differentiate and reconstitute the injured mouse recepient liver. The main a...