This work was supported by the National Institute of Health grant R24OD018546 (to L.Y.P. and S.G.). The authors would like to thank Weizhe Li, Ph.D., for the help in surgical procedures, Amanda Branch Woods, B.S., Yan Cheng for immunohistology, UNMC flow cytometry research facility members Director Phillip Hexley, Ph.D., Victoria B. Smith, B.S., and Samantha Wall, B.S., UNMC advanced microscopy core facility members Janice A. Taylor, B.S., and James R. Talaska, B.S., for the technical support. The authors acknowledge Drs. Mamoru Ito and Hiroshi Suemizu from CIEA for providing TK-NOG mice and Dr. Joachim Baumgart for providing treosulfan. The authors thank Dr. Adrian Koesters, UNMC, for her editorial contribution to the manuscript.

This protocol has been approved by the Institutional Animal Care and Use Committee (IACUC)&#160;at the University of Nebraska Medical Center.
NOTE: Obtain approval from the local IACUC before performing experiments on animals.
1. Processing of Umbilical Cord Blood and the Isolation of Human HSPCs
<ol>
	<li>Perform all steps of the protocol under sterile conditions in laminar flow cabinets.</li>
	<li>Take umbilical cord blood (CB) collected in hepa...

<ol>
	<li>Smith, 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=Factors+associated+with+specific+causes+of+death+amongst+HIV-positive+individuals+in+the+D:A:D+Study.">Factors associated with specific causes of death amongst HIV-positive individuals in the D:A:D Study.</a> AIDS. 24 (10), 1537-1548 (2010).</li><li>Puoti, 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=Mortality+for+liver+disease+in+patients+with+HIV+infection:+a+cohort+study.">Mortality for liver disease in patients with HIV infection: a cohort study.</a> Journal of Acquired Immune Deficiency Syndromes. 24 (3), 211-217 (2000).</li><li>Rodriguez-Mendez, M. 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F., 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=Hepatitis+B+in+patients+with+HIV+infection:+relationship+to+AIDS+and+patient+survival.">Hepatitis B in patients with HIV infection: relationship to AIDS and patient survival.</a> Annals of Internal Medicine. 117 (10), 837-838 (1992).</li><li>Lacombe, K., Rockstroh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+and+viral+hepatitis+coinfections:+advances+and+challenges.">HIV and viral hepatitis coinfections: advances and challenges.</a> Gut. 61, 47-58 (2012).</li><li>Brehm, M. A., Jouvet, N., Greiner, D. L., Shultz, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mice+for+the+study+of+infectious+diseases.">Humanized mice for the study of infectious diseases.</a> Current Opinion in Immunology. 25 (4), 428-435 (2013).</li><li>Billerbeck, E., 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=Humanized+mice+efficiently+engrafted+with+fetal+hepatoblasts+and+syngeneic+immune+cells+develop+human+monocytes+and+NK+cells.">Humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and NK cells.</a> The Journal of Hepatology. 65 (2), 334-343 (2016).</li><li>Dagur, R. 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=Human+hepatocyte+depletion+in+the+presence+of+HIV-1+infection+in+dual+reconstituted+humanized+mice.">Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice.</a> Biology Open. 7 (2), (2018).</li><li>Gaska, J. M., Ploss, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+viral+pathogenesis+in+humanized+mice.">Study of viral pathogenesis in humanized mice.</a> Current Opinion in Virology. 11, 14-20 (2015).</li><li>Gorantla, S., Poluektova, L., Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+HIV-associated+neurocognitive+disorders.">Rodent models for HIV-associated neurocognitive disorders.</a> Trends in Neurosciences. 35 (3), 197-208 (2012).</li><li>Xu, 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=Chimeric+TK-NOG+mice:+a+predictive+model+for+cholestatic+human+liver+toxicity.">Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity.</a> The Journal of Pharmacology and Experimental Therapeutics. 352 (2), 274-280 (2015).</li><li>Washburn, M. L., 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=A+humanized+mouse+model+to+study+hepatitis+C+virus+infection,+immune+response,+and+liver+disease.">A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease.</a> Gastroenterology. 140 (4), 1334-1344 (2011).</li><li>Gutti, T. L., 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+hepatocytes+and+hematolymphoid+dual+reconstitution+in+treosulfan-conditioned+uPA-NOG+mice.">Human hepatocytes and hematolymphoid dual reconstitution in treosulfan-conditioned uPA-NOG mice.</a> The American Journal of Pathology. 184 (1), 101-109 (2014).</li><li>Strick-Marchand, 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=A+novel+mouse+model+for+stable+engraftment+of+a+human+immune+system+and+human+hepatocytes.">A novel mouse model for stable engraftment of a human immune system and human hepatocytes.</a> PLoS One. 10 (3), 0119820 (2015).</li><li>Keng, C. 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=Characterisation+of+liver+pathogenesis,+human+immune+responses+and+drug+testing+in+a+humanised+mouse+model+of+HCV+infection.">Characterisation of liver pathogenesis, human immune responses and drug testing in a humanised mouse model of HCV infection.</a> Gut. 65 (10), 1744-1753 (2016).</li><li>Li, F., Nio, K., Yasui, F., Murphy, C. 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L., Kovalev, G. 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The liver is compromised and damaged in HIV-infected patients24. Experimental small animal models for studying human liver diseases in the presence of HIV-1 is extremely limited, despite the availability of a few cotransplanted animal models with CD34+ HSPCs and hepatocytes7,12,25. In in vitro experiments, hepatocytes are shown to have low-level HIV-1 infection26. Humani...

Since the advent of antiretroviral therapy, there has been a substantial decrease in deaths related to HIV-1 monoinfection. However, liver disease has emerged as a common cause of morbidity in HIV-infected patients1,2. Coinfections of hepatitis viruses with HIV-1 infection are more common, accounting for 10% - 30% of HIV-infected persons in the United States3,4,5.
The host-specificity of HIV-1 and hepatitis viruses limits the utility of small animal models to study human-specific infectious diseases or to investigate multiple aspects of HIV-1-associated liver pathogenesis. Immunodeficient mice that permit the engraftment of human cells and/or tissues (termed humanized mouse models) are acceptable animal models for preclinical studies6,7,8. Since the introduction of humanized mice in the early 2000s, multiple preclinical studies of cholestatic human liver toxicity, human-specific pathogens, including HIV-1 and HIV-associated neurocognitive disorders, Epstein Barr virus, hepatitis, and other infectious diseases, have been investigated in these mice6,9,10,11. Multiple mouse models for CD34+ HSPCs and/or human hepatocyte transplantation have long been developed and have improved over time to study the disease pathogenesis of Hepatitis B virus (HBV)-associated liver disease12,13,14. Several models for HSPC and human hepatocyte (HEP) transplantation are based on strains, known as NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac)8,13, NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)15, Balb/C-Rag2-/- &#947;c-/- (Rag2tm1.1Flv Il2rgtm1.1Flv/J)12, and fah-/- NOD rag1-/-il2r&#947;null mouse16. However, each model has its own advantages and limitations; for example, AFC8 dual humanized mice for HEPs and human stem cells (HSCs) on a Balb/C-Rag2-/- &#947;c-/-&#160;background enables the successful engraftment of immune cells and HSCs, but there is an absence of an antigen-specific T- and B-cell response in this model12. The major concerns in reconstituting double humanized mice include suboptimal engraftment, a lack of suitable models to support different tissues, mismatched conditions, immune rejection, or graft-versus-host disease (GVHD), and technical difficulties, such as risky manipulations with newborns and high mortality rates due to metabolic abnormalities13.
Although humanized mice have been used for HIV research for many years17,18,19, the use of humanized mice to study liver damage caused by HIV-1 has been limited20. We previously reported the establishment of a dual humanized TK-NOG mouse model and its application in HIV-associated liver disease8. This model shows the robust engraftment of liver and immune cells and recapitulates HIV infection pathogenesis. This discussion presents a detailed protocol, including the most critical steps in the transplantation of human hepatocytes. A description of the HSPCs required for a successful engraftment of HEPs and the establishment of a functional immune system in TK-NOG mice is also presented. The use of these mice to study HIV-associated liver immunopathogenesis is detailed. TK-NOG male mice carrying a liver-specific herpes simplex virus type 1 thymidine kinase (HSV-TK) transgene are used. Mouse liver cells expressing this transgene can easily be ablated after a brief exposure to a nontoxic dose of GCV. Transplanted human liver cells are stably maintained within the mouse liver without exogenous drugs21. The mice are also preconditioned with nonmyeloablative doses of treosulfan to create a niche in the mouse bone marrow for human cells8. Immunodeficient TK-NOG mice are intrasplenically injected with HEPs and multipotent HSPCs. The mice are then regularly monitored for blood and liver reconstitution by blood immunophenotyping and measurements of serum human-albumin levels, respectively. Mice with a successful reconstitution of more than 15% for both human immune cells and HEPs are intraperitoneally injected with HIV-1. The effect of HIV on the liver can be assessed as early as 4 - 5 weeks postinfection. It is critical to note that, because HIV-1 is used, all necessary precautions must be taken while handling the virus and injecting it into mice.

<table border="0" cellpadding="0" cellspacing="0" style="width:553px;" width="552">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">27G1/2&#34; needles</td>
			<td style="width:135px;">BD biosciences</td>
			<td style="width:127px;">305109</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:125px;">30G1/2&#34; needles</td>
			<td style="width:135px;">BD biosciences</td>
			<td style="width:127px;">305106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">5 mL polystyrene&#160; round-bottom tube 12 x 75 mm style</td>
			<td style="width:135px;">Corning</td>
			<td style="width:127px;">352054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">BD 1 mL Tuberculin Syringe Without Needle</td>
			<td style="width:135px;">BD biosciences</td>
			<td style="width:127px;">309659</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">BD FACS array bioanalyzer&#160;</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">For purity check of eluted CD34+ cells&#160;</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:125px;">BD FACS array software</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Software to analysis acquired CD34+ cell on FACS array</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">BD FACS lysing solution</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">349202</td>
			<td style="width:167px;">To lyse red blood cells</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">BD LSR II</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Instrument for acquisiton of flow cytometry samples</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">BD&#160;Vacutainer Plastic Blood Collection Tube</td>
			<td style="width:135px;">BD biosciences</td>
			<td style="width:127px;">BD 367874</td>
			<td style="width:167px;">To collect Cord blood</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Bovine Serum Albumin&#160;</td>
			<td style="width:135px;">Sigma-aldrich</td>
			<td style="width:127px;">A9576</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Buprenorphine</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Controlled substance and pain-killer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD14-PE</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">555398</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD19-BV605</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">562653</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:125px;">CD34 MicroBead Kit, human</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-046-702</td>
			<td style="width:167px;">For isoation of&#160;&#160; CD34+ HSPC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">CD34-PE, human</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-081-002</td>
			<td style="width:167px;">Antibody used for purity check of eluted CD34+ cells&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD3-AF700</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">557943</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD45-PerCPCy5.5</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">564105</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD4-APC</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">555349</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">CD8-BV421</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">562428</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cell counting slides</td>
			<td style="width:135px;">Bio-rad</td>
			<td style="width:127px;">1450015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">ChargeSwitch gDNA Mini Tissue Kit</td>
			<td style="width:135px;">Thermofisher scientific</td>
			<td style="width:127px;">CS11204</td>
			<td style="width:167px;">for extraction of genomic DNA from ear piece</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cobas Amplicor system v1.5&#160;</td>
			<td style="width:135px;">Roche Molecular Diagnostics</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">bioanalyzer to measure viral load</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cotton-tipped applicators&#160;</td>
			<td style="width:135px;">&#160;McKesson</td>
			<td style="width:127px;">24-106-2S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Cytokeratin-18 (CK18)</td>
			<td style="width:135px;">DAKO</td>
			<td style="width:127px;">M7010</td>
			<td style="width:167px;">Specific to human</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:135px;">Sigma-aldrich</td>
			<td style="width:127px;">D2650-5X5ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:125px;">Extension set Microbore Slide Clamp(s) Fixed Male Luer Lock. L: 60 in L: 152 cm PV: 0.55 mL Fluid Path Sterile</td>
			<td style="width:135px;">BD biosciences</td>
			<td style="width:127px;">30914</td>
			<td style="width:167px;">Attached to dispensing pippet and to load with HSPC and HEP suspesion</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">FACS Diva version 6</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">flow cytometer software required for&#160; acqusition of sample</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:127px;">10438026</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">FLOWJO analysis software 
			v10.2</td>
			<td style="width:135px;">FLOWJO, LLC</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">flow cytometry analysis software</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Ganciclovir</td>
			<td style="width:135px;">APP Pharmaceuticals, Inc.</td>
			<td style="width:127px;">315110</td>
			<td style="width:167px;">Prescripition drug</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Greiner MiniCollect EDTA Tubes</td>
			<td style="width:135px;">Greiner bio-one</td>
			<td style="width:127px;">450475</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Hepatocytes thawing medium&#160;</td>
			<td style="width:135px;">Triangle Research Labs&#160;</td>
			<td style="width:127px;">MCHT50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Horizon Open Ligating Clip Appliers</td>
			<td style="width:135px;">Teleflex</td>
			<td style="width:127px;">537061</td>
			<td style="width:167px;">To hold the ligating clips</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Hospira Sterile Water for Injection</td>
			<td style="width:135px;">ACE surgical supply co. Inc.</td>
			<td style="width:127px;">001-1187</td>
			<td style="width:167px;">For dilution of Buprenorphine (pain-killer)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Human Albumin ELISA Quantitation Set</td>
			<td style="width:135px;">Bethyl laboratories</td>
			<td style="width:127px;">E80-129</td>
			<td style="width:167px;">For assesing human albumin levels in mouse serum</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Human hepatocyte</td>
			<td style="width:135px;">Triangle Research Labs&#160;</td>
			<td style="width:127px;">HUCP1</td>
			<td style="width:167px;">&#160;Cryopreserved human hepatocytes, induction qualified&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Iris Scissors, Straight</td>
			<td style="width:135px;">Ted Pella, Inc.</td>
			<td style="width:127px;">13295</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">Lancet</td>
			<td style="width:135px;">MEDIpoint</td>
			<td style="width:127px;">Goldenrod 5 mm</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:125px;">LS columns&#160;</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-042-401</td>
			<td style="width:167px;">Used to entrap CD34+ microbeads (positive selection)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Lymphocyte Separation Medium (LSM)</td>
			<td style="width:135px;">MP Biomedicals</td>
			<td style="width:127px;">50494</td>
			<td style="width:167px;">For isoation of&#160;&#160; lymphocytes from peripheral blood</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">MACS MultiStand</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-042-303</td>
			<td style="width:167px;">holds Qudro MACS seperator and LS columns</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">McPherson-Vannas Micro Dissecting Spring Scissors</td>
			<td style="width:135px;">Roboz Surgical Instrument Co.</td>
			<td style="width:127px;">RS-5605</td>
			<td style="width:167px;">Used to make an incision on skin to expose spleen</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Micro Dissecting Forceps</td>
			<td style="width:135px;">Roboz Surgical Instrument Co.</td>
			<td style="width:127px;">RS-5157&#160;</td>
			<td style="width:167px;">to hold and pull out spleen from peritoneal cavity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">mouse CD45-FITC</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:127px;">553080</td>
			<td style="width:167px;">mouse-specific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">PBS (Phosphate Buffered Saline)</td>
			<td style="width:135px;">Hyclone</td>
			<td style="width:127px;">SH30256.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">Qudro MACS separator&#160;</td>
			<td style="width:135px;">Miltenyi Biotec</td>
			<td style="width:127px;">130-090-976</td>
			<td style="width:167px;">holds four LS columns</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">RPMI 1640 medium</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:127px;">11875093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">StepOne Plus Real Time PCR&#160;</td>
			<td style="width:135px;">Applied Biosystems</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Instrument used&#160; to&#160; genotype</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">Stepper Series Repetitive Dispensing Pipette 1ml</td>
			<td style="width:135px;">DYMAX CORP</td>
			<td style="width:127px;">T15469</td>
			<td style="width:167px;">Used to&#160; dispense&#160; HSPC and HEP supension in controlled manner</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Suturevet PGA synthetic absorbale suture</td>
			<td style="width:135px;">Henry Schein Animal Health</td>
			<td style="width:127px;">41178</td>
			<td style="width:167px;">Suturing of skin and peritoneum</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:135px;">Thermofisher scientific</td>
			<td style="width:127px;">4369016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:125px;">TC20 automated cell counter</td>
			<td style="width:135px;">Bio-rad</td>
			<td style="width:127px;">1450102</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:125px;">TK-NOG mice&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Provided by the Central Institute for Experimental Animals (CIEA, Japan; Drs. Mamoru Ito and Hiroshi Suemizu)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Treosulfan</td>
			<td style="width:135px;">Medac GmbH</td>
			<td style="width:127px;"></td>
			<td style="width:167px;">Provided by &#160;Dr. Joachim Baumgart (medac GmbH)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">Trypan Blue</td>
			<td style="width:135px;">Bio-rad</td>
			<td style="width:127px;">1450022</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:125px;">Vannas-type Micro Scissors, Straight, 80mm L</td>
			<td style="width:135px;">Ted Pella, Inc.</td>
			<td style="width:127px;">1346</td>
			<td style="width:167px;">Used to make an incision on skin to expose spleen</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:125px;">Weck hemoclip traditional titanium ligating clips</td>
			<td style="width:135px;">Esutures</td>
			<td style="width:127px;">523700</td>
			<td style="width:167px;">To ligate the spleen post-injection</td>
		</tr>
	</tbody>
</table>

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.

The establishment of a dual humanized mouse model with human liver and immune cells can be easily monitored at each step with very simple ELISA and flow cytometry, respectively. Flow cytometry is regularly performed to evaluate the development of a functional immune system and to see the effect of HIV infection on immune cells. In dual humanized mice, the development of functional immune cells can range from 15% to 90% of the lymphocyte gate. Representative subsets of immune cells are sho...

Watch this Scientific Journal Video about Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis at JoVE.com

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

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 ...

The overall aim of the protocol is to generate a humanized mouse model with functional human immune system and liver using human hematopoietic stem cells and hepato site. This method can help answer key questions in genetic liver humanized mice. This method provides a powerful tool to study immunopathology caused by HIV as observed in people.Demonstrating the process will be by Yimin Sun, a supervisor from the pathology and microbiology department, along with Weimin Wang and Edwrd Makarov, research technologist. To begin, transfer umbilical cord blood collected in heparinized tubes to a sterile laminar flow cabinet. Add PBS until the volume is increased to 35 milliliters.Layer the sample on top of lymphocyte separation medium and centrifuge it 400 times G and at four degrees celsius for 35 minutes with no breaks. Next, carefully remove the top plasma layer and use a transfer pipette to transfer the white buffy coat interface to a new tube. Re-suspend the buffy coat in 30 to 40 milliliters of ice cold buffer.Using a pipette, combine 20 microliters of the cell suspension with 20 microliters of 0.4%trypan blue. Pipette 10 microliters of this mixture into the outer opening of either of the two chambers of a counting slide, and insert the slide into an automated cell counter to count the cells. Centrifuge the cells at 300 times G and at four degrees celsius for 10 minutes.Carefully aspirate the supernatant and add 300 microliters of ice cold buffer. Add 100 microliters of human FC receptor blocking reagent and 100 microliters of monoclonal mouse anti-human CD34 antibody conjugated MicroBeads for up to 100 million cells. Incubate for 30 minutes at four degrees celsius.Add 10 milliliters of ice cold buffer to wash the cells and centrifuge it 300 times G and four degrees celsius for 10 minutes. Carefully remove the supernatant and re-suspend the pellet in 500 microliters of ice cold buffer. After this, place a positive selection LS column in the magnetic activated cell sorting field and pass it through with three milliliters of ice cold buffer.Load the sample into the LS column that can entrap MicroBeads bound to human CD34 positive cells in samples, and allow it to flow under the influence of gravity into the collection tube. Wash the column three times with ice cold buffer and collect the eluent in the same collection tube. Then, plunge the column with five milliliters of ice cold buffer to allude the CD34 positive cells into a new collection tube.Repeat the procedure to achieve a purity of over 90%Next, use trypan blue dye and a hemocytometer to count the eluded CD34 positive cells. After counting, centrifuge the cells at 300 times G for five minutes. Discard the supernatant and re-suspend the cells in 125 microliters of PBS for an injection to be used immediately in transplantation.To check the purity of the CD34 positive eluent, take 50 microliters of the suspension and incubate it with 10 microliters of PE conjugated anti-human CD34 antibody at four degrees celsius for 30 minutes. After this, wash and re-suspend the cells in PBS and proceed to perform flow cytometry. After acquisition, analyze the data as outlined in the text protocol.First, retrieve and thaw the cryopreserved hepatocytes as outlined in the text protocol. Then, remove the bio cap and pour the thawed hepatocytes into a 50 milliliter conical tube containing warmed thawing medium. Rock the tube by hand for a few seconds to suspend the cells.Pellet the cells at 100 times G and at room temperature for eight minutes. Wash the pelleted cells in PBS with 0.1%BSA and pool them with either fresh or thawed HSPCs in PBS to a final volume of 80 microliters per mouse. First, attach one end of a sterile extension tube to a 30 gauge needle and the other end to a one milliliter syringe.Fill the syringe with the suspension of pooled HEPs and HSPCs. Fit the syringe in the notch of a repetitive dispensing pipette and adjust the dispenser to dispense 10 microliters in each press. Shave each mouse as outlined in the text protocol and scrub the left side of the body of each mouse with 10%povidone iodine followed 70%isopropyl alcohol.Using Vannas type scissors, make an small incision in the skin, muscle and peritoneum at the left of the peritoneal wall to enter the peritoneal cavity approximately 5 millimeters below the lower edge of the rib cage. Locate the spleen and use forceps to pull it slightly to the operating area for easy access and insert the 30 gauge needle into the lower pole of the spleen. Next, unlock the plunger of the dispensing pipette and dispense 10 microliters of the volume at a time, with a limit of 60 to 80 microliters per spleen.Retract the needle slowly and clip the spleen with ligating clips using a ligation applier. Then, use cotton-tipped applicators wetted with sterile PBS to push the spleen back into the body cavity. Use an interrupted suture pattern to close the muscle layer of the abdominal wall.Accomplish skin closure with an interrupted suture pattern using non-absorbable sutures. Transfer all needed materials to a designated Biosafety Level Two Plus facility. Wear personal protection equipment including a disposable cover all gown, shoe covers, a face mask and double gloves, including cut resistant gloves, at all times while working with the virus.Select mice with a reconstitution of more than 15%of human CD45 positive cells and with a presence of human albumin in the serum for HIV-1 infection. Intraperitoneally inject the mice with 1, 000 to 10, 000 tissue culture infectious doses of 50 HIV-1 ADA in a volume of 100 to 200 microliters per mouse. After euthanizing the mice, excise the liver from each mouse as outlined in the text protocol.Collect and fix the livers in 4%paraformaldehyde overnight. The establishment of a dual humanized mouse model with human liver and immune cells can be easily monitored at each step with very simple ELIZA and flow cytometry, respectively. Flow cytometry is regularly performed to evaluate the development of a functional immune system and to see the effect of HIV infection on immune cells.In dual humanized mice, the development of functional immune cells can range from 15 to 90%of the lymphocyte gate. For the evaluation of the engraftment of human hepatocytes, ELIZA for human-specific albumin levels is performed monthly on mouse serum. Mice engrafted with both HSPCs and HEPs show human-specific albumin levels ranging from approximately seven micrograms per milliliter to 377 micrograms per milliliter at one month, continuing grow over the time of observation.The effect of HIV infection on human immune cells in the blood of dual humanized mice is monitored by flow cytometry and on HEPs in the liver by human-specific albumin ELIZA. By five weeks, HIV-1 causes a decrease in human albumin levels in the serum and there is a depletion of human CK18 positive hepatocytes in the liver sections of dual humanized mice. A lower of ratio of CD4 to CD8 is typically observed in the blood and liver of HIV-infected mice, compared to levels noted in the same mouse before infection.Blood should be layered carefully on top of lymphocyte separation medium for isolations of buffy coat. For the surgery, hold the spleen gently and insert the needle in lower pole of the spleen. Following the isolation of hematopoietic stem cells, it is crucial to check the purity of CD34 positive hematopoietic stem cells to avoid CD3 positive cells and acute injections of hepatocyte from different donor.As the humanized mice show physiological aspect of human immune system and liver, the developed mice could be utilized to study physiopathogenesis of HIV and physical infections and cirrhosis.

establishment-dual-humanized-tk-nog-mouse-model-for-hiv-associated

Research

JoVE Journal

Immunology and Infection

8.4K 조회수.  University of Nebraska Medical Center. 프로토콜의 전반적인 목표는 인간 조혈 줄기 세포 및 헤파토 부위를 사용하여 기능적 인간 면역 체계및 간을 가진 인간화된 마우스 모델을 생성하는 것입니다. 이 방법은 유전 간 인간화 마우스에 있는 중요한 질문에 대답하는 것을 도울 수 있습니다. 이 방법은 사람들에서 관찰된 바와 같이 HIV에 기인한 면역 병리학을 공부하는 강력한 공구를 제공합니다.이 과정을 시?...