Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

Abstract

Source: Lanis, J. M., et al. Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors. J. Vis. Exp. (2022)

This video demonstrates a technique for generating human immune system (HIS) mice. CD34⁺ hematopoietic stem cells (HSCs) are isolated from human cord blood buffy coat cells using antibody-coated magnetic beads. The CD34⁺ HSCs are expanded in a growth medium enriched with cytokines and cryopreserved. Finally, the thawed HSCs are injected into an immunodeficient mouse pup to establish the HIS mouse model.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Generation of HIS mice

1. Mouse husbandry of BALB/c -Rag2^null Il2RγC^null Sirpα^NOD (BRGS) mice
   NOTE: This strain is extremely immunodeficient, with no T, B, or natural killer (NK) cells. Therefore, rigorous measures must be used to prevent opportunistic infections. Maintain the colony....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL syringe w/needles	McKesson	1031815
15 mL tubes	Grenier Bio-One	188271
2-mercaptoethanol	Sigma	M6250
50 mL tubes	Grenier Bio-One	227261
AutoMACS Pro Separator	Miltenyi	130-092-545
BSA	Fisher Scientific	BP1600100
Chill 15 Rack	Miltenyi	130-092-952
Embryonic Stemcell FCS	Gibco	10439001
Eppendorf Tubes; 1.5 mL volume	Grenier Bio-One	616201
Excel	Microsoft
FBS	Benchmark	100-106 500mL
Ficoll Hypaque	GE Healthcare	45001752
glass pipettes	DWK Life Sciences	63A53
HBSS w/ Ca & Mg	Sigma	55037C
MultiRad 350	Precision X-Ray
PBS	Corning	45000-446
PRISM	Graphpad
Rec Hu FLT3L	R&D systems	308-FK-005/CF
Rec Hu IL6	R&D systems	206-IL-010/CF
Rec Hu SCF	R&D systems	255SC010
Serological pipettes 25 mL	Fisher Scientific	1367811
Sterile filter	Nalgene	567-0020
Sterile molecular water	Sigma	7732-18-5
Yeti Cell Analyzer	Bio-Rad	12004279