Developing a Humanized Mouse Model with Human Liver Cells and Immune Cells

Abstract

Source: Dagur, R. S. et al., Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis. J. Vis. Exp. (2019)

This video demonstrates a method for generating a dual humanized mouse model with a human immune system and liver cells. It involves transplanting human liver cells and hematopoietic stem and progenitor cells into a transgenic immunodeficient mouse via intrasplenic injection, leading to engraftment of liver cells and development of human immune cells.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Processing of Umbilical Cord Blood and the Isolation of Human Hematopoietic Stem/Progenitor Cells (HSPCs) Perform all steps of the protocol under sterile conditions in laminar flow cabinets. Take umbilical cord blood (CB) collected in heparinized tubes and make the volume up to 35 mL by adding phosphate-buffer…

Representative Results

Figure 1: Schematic of the enrichment of CD34+ cells from cord blood. (A) Cord blood is layered on lymphocytes separation medium (LSM) and centrifuged to isolate the buffy coat. (B) LS columns are placed on a magnetic stand and rinsed with a BSA buffer, followed by adding a buffy coat. Cells positive for CD34 are trapped in the columns, and C…

Materials

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
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
Cell counting slides	Bio-rad	1450015
ChargeSwitch gDNA Mini Tissue Kit	Thermofisher scientific	CS11204	For extraction of genomic DNA from ear piece
Cotton-tipped applicators	McKesson	24-106-2S
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
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
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