Name: generation of a humanized mouse liver using human hepatic stem cells
Uploaded: 2016-08-29T15:00:00
Description: Watch this Scientific Journal Video about Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells at JoVE.com

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>
	<li>Muruganandan, S., Sinal, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+as+clinically+relevant+models+for+the+study+of+cytochrome+P450-dependent+metabolism.">Mice as clinically relevant models for the study of cytochrome P450-dependent metabolism.</a> Clin. Pharmacol. Ther. 83, 818-828 (2008).</li><li>Nishimura, T., 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=Using+chimeric+mice+with+humanized+livers+to+predict+human+drug+metabolism+and+a+drug-drug+interaction.">Using chimeric mice with humanized livers to predict human drug metabolism and a drug-drug interaction.</a> J. Pharmacol. Exp. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replacement+of+diseased+mouse+liver+by+hepatic+cell+transplantation.">Replacement of diseased mouse liver by hepatic cell transplantation.</a> Science. 263, 1149-1152 (1994).</li><li>Haridass, D., 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=Repopulation+efficiencies+of+adult+hepatocytes,+fetal+liver+progenitor+cells,+and+embryonic+stem+cell-derived+hepatic+cells+in+albumin-promoter-enhancer+urokinase-type+plasminogen+activator+mice.">Repopulation efficiencies of adult hepatocytes, fetal liver progenitor cells, and embryonic stem cell-derived hepatic cells in albumin-promoter-enhancer urokinase-type plasminogen activator mice.</a> Am. J. Pathol. 175, 1483-1492 (2009).</li><li>Sharma, A. D., 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=Murine+embryonic+stem+cell-derived+hepatic+progenitor+cells+engraft+in+recipient+livers+with+limited+capacity+of+liver+tissue+formation.">Murine embryonic stem cell-derived hepatic progenitor cells engraft in recipient livers with limited capacity of liver tissue formation.</a> Cell. Transplant. 17, 313-323 (2008).</li><li>Peltz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+'humanized'+mice+improve+drug+development+in+the+21st+century.">Can 'humanized' mice improve drug development in the 21st century.</a> Trends Pharmacol. Sci. 34, 255-260 (2013).</li><li>Zhu, S., 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=Mouse+liver+repopulation+with+hepatocytes+generated+from+human+fibroblasts.">Mouse liver repopulation with hepatocytes generated from human fibroblasts.</a> Nature. 508, 93-97 (2014).</li><li>Saito, 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=Diphtheria+toxin+receptor-mediated+conditional+and+targeted+cell+ablation+in+transgenic+mice.">Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice.</a> Nat. Biotechnol. 19, 746-750 (2001).</li><li>Ishii, T., 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=Transplantation+of+embryonic+stem+cell-derived+endodermal+cells+into+mice+with+induced+lethal+liver+damage.">Transplantation of embryonic stem cell-derived endodermal cells into mice with induced lethal liver damage.</a> Stem Cells. 25, 3252-3260 (2007).</li><li>Machimoto, T., 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=Improvement+of+the+survival+rate+by+fetal+liver+cell+transplantation+in+a+mice+lethal+liver+failure+model.">Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model.</a> Transplantation. 84, 1233-1239 (2007).</li><li>Taniguchi, H., Zheng, Y. -. 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. Ther. 6, 1-12 (2015).</li><li>Strom, S. C., Davila, J., Grompe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Mice+with+Humanized+Liver:+Tools+for+the+Study+of+Drug+Metabolism,+Excretion,+and+Toxicity.">Chimeric Mice with Humanized Liver: Tools for the Study of Drug Metabolism, Excretion, and Toxicity.</a> Hepatocytes. , 491-509 (2010).</li><li>Zhang, D., Luo, G., Ding, X., Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+experimental+models+of+drug+metabolism+and+disposition+in+drug+discovery+and+development.">Preclinical experimental models of drug metabolism and disposition in drug discovery and development.</a> Acta Pharm. Sin. 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. Biol. Chem. 270, 1015-1019 (1995).</li><li>Liu, Y., Yang, R., He, Z., Gao, W. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+organs+from+stem+cells.">Generation of functional organs from stem cells.</a> Cell Regener. 2, 1 (2013).</li></ol>

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

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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 ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Measurement of the Hepatic Venous Pressure Gradient and Transjugular Liver Biopsy

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced chronic liver disease followed by an instruction for transjugular biopsy. Under local anesthesia and ultrasound guidance, a catheter introducer sheath is placed in the right internal jugular vein. Using&#160;fluoroscopic guidance, a balloon catheter is advanced into the inferior vena cava (IVC) and inserted into a large hepatic vein. Correct and sufficient wedge position of the catheter is ensured by injecting contrast media while the balloon is blocking the outflow of the cannulated hepatic vein. After calibrating the external pressure transducer, continuous pressure recordings are obtained with triplicate recordings of the wedged hepatic venous pressure (WHVP) and free hepatic venous pressure (FHVP). The difference between FHVP and WHVP is referred to as HVPG, with values &#8805;10 mm Hg indicating clinically significant portal hypertension (CSPH). Before removing the catheter, pressure readings obtained in the IVC at the same level, as well as the right atrial pressure are recorded.
Finally, a transjugular liver biopsy can be obtained via the same vascular route. Different systems are available; however, core biopsy needles are preferred over aspiration needles, especially for cirrhotic livers. Again, under fluoroscopic guidance a biopsy needle introducer sheath is advanced into an hepatic vein. Next, the transjugular biopsy needle is gently advanced through the introducer sheath: (i) in case of aspiration biopsy, the needle is advanced into the liver parenchyma under aspiration and then removed quickly, or (ii) in case of a core biopsy, the cutting-mechanism is triggered inside the parenchyma. Several separate passages can be safely performed to obtain sufficient liver specimens via transjugular biopsy. In experienced hands, the combination of these procedures takes about 30-45 min.

Here, we present a protocol for measurement of hepatic venous pressure gradient (HVPG),the gold standard to diagnose clinically significant portal hypertension. Moreover, we describe how to perform a transjugular liver biopsy within the same session.

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...