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

Summary

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.

Abstract

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'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-Prkdc^scid Il2rg^tm1Sug 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.

Introduction

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 patients^1,2. Coinfections of hepatitis viruses with HIV-1 infection are more common, accounting for 10% - 30% of HIV-infected persons in the United States^3,4,5.

The host-specificity of HIV-1 and hepatitis viruses limits the utility of small animal models to study human-specific infectious....

Protocol

This protocol has been approved by the Institutional Animal Care and Use Committee (IACUC) 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

1. Perform all steps of the protocol under sterile conditions in laminar flow cabinets.
2. Take umbilical cord blood (CB) collected in hepa.......

Discussion

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 hepatocytes^7,12,25. In in vitro experiments, hepatocytes are shown to have low-level HIV-1 infection²⁶. Humani.......

Materials

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