TABLE 2.

Important Considerations for Using Humanized Mice in PSC Immunogenicity Studies

Experimental Considerations	Mouse Model Considerations
Immunogenicity assays	Immunodeficient mouse strain
HLA typing of PSCs, humanization donor tissues	Utilization of proper immunodeficient strain and humanized model type (PBL-hu, HSC-hu, BLT type, NeoThy, Other)
Transplant timing development of tissues in vitro	Utilizing standard humanized mice strains vs genetically modified strains
Optimal experimental window duration Time for graft immune infiltration/other considerations	Threshold for human chimerism ≥20% human CD45+ ≥2% human CD3+ for T cell based studies
Pair in vivo humanized mice models with complementary in vitro assays	Timeframe for assaying human chimerism At minimum: start of study (e.g., for NeoThy mice @ 12–16 weeks post-humanization) and end of study
Source of PSCs iPS vs ES Genetically modified PSC lines	Consult literature to stay up-to-date on best models available
Optimal graft preparation: 2D vs 3D	First-generation immunodeficient mice strains (e.g., CB17 SCID) are not recommended for most applications
In vivo environment/transplant site best suited to study tissue type of interest	Benefits of novel mouse models vs feasibility to use/body of reference literature
Transplantation format best suited to experimental questions	Myeloablation via irradiation vs chemical vs genetic manipulation of c-kit?
Characterization of cell populations present in cell preparations (purity)	PBL-hu for short-term studies or those with limited access to HSPCs and/or thymus tissue
Pre- and post-transplant functional characterization of grafts	BLT or NBSGW for PSC studies if at all possible