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Generation of the NeoThy mouse model for human immune system studies


Humanized mouse models, created via transplantation of human hematopoietic tissues into immune-deficient mice, support a number of research applications, including transplantation immunology, virology and oncology studies. As an alternative to the bone marrow, liver, thymus humanized mouse, which uses fetal tissues for generating a chimeric human immune system, the NeoThy humanized mouse uses nonfetal tissue sources. Specifically, the NeoThy model incorporates hematopoietic stem and progenitor cells from umbilical cord blood (UCB) as well as thymus tissue that is typically discarded as medical waste during neonatal cardiac surgeries. Compared with fetal thymus tissue, the abundant quantity of neonatal thymus tissue offers the opportunity to prepare over 1,000 NeoThy mice from an individual thymus donor. Here we describe a protocol for processing of the neonatal tissues (thymus and UCB) and hematopoietic stem and progenitor cell separation, human leukocyte antigen typing and matching of allogenic thymus and UCB tissues, creation of NeoThy mice, assessment of human immune cell reconstitution and all experimental steps from planning and design to data analysis. This entire protocol takes a total of ~19 h to complete, with steps broken up into multiple sessions of 4 h or less that can be paused and completed over multiple days. The protocol can be completed, after practice, by individuals with intermediate laboratory and animal handling skills, enabling researchers to make effective use of this promising in vivo model of human immune function.

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Fig. 1: Impact of immune-deficient mouse host strain on human chimerism.
Fig. 2: HLA matching of allogeneic thymus and UCB HSPCs.
Fig. 3: Comparison of human immune cell chimerism in three immune-deficient mouse strains.
Fig. 4: Neonatal thymus processing.
Fig. 5: NeoThy humanization surgery.
Fig. 6: Mouse retro-orbital blood collection.
Fig. 7: Flow cytometry engraftment analysis.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.


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This study was supported in part by the Wisconsin Alumni Research Foundation, NIH NIAID 75N93021C00004, NIH NHLBI U01HL134764, NIH NHLBI 156HL16518901 the UW Carbone Cancer Center Support Grant P30 CA014520 (M.E.B.), CA034196 (L.D.S.), AI132963 (M.A.B. and L.D.S.), OD026440 (M.A.B. and L.D.S.) and NIDDK-supported Human Islet Research Network (HIRN, DK104218 (M.A.B.). We thank J. Zellner and D. Maya for manuscript editing assistance, and K. Howard for helpful discussion.

Author information

Authors and Affiliations



N.M.D.R. and L.H. helped to write the manuscript, and performed and analyzed experiments; L.M., J.S.B. and C.M.D. assisted with manuscript text and performed experiments; W.J.H. analyzed data and assisted with manuscript preparation; J.G.K., M.A.B. and L.D.S. performed experiments, analyzed data and assisted with manuscript preparation; M.E.B. supervised and performed experiments, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Matthew E. Brown.

Ethics declarations

Competing interests

M.E.B. is a consultant for Taconic Biosciences; L.D.S. is a consultant for Dren Bio and Blue Rock Therapeutics; M.A.B. is a consultant for The Jackson Laboratory. The other authors declare no competing interests.

Peer review

Peer review information

Lab Animal thanks Larisa Poluektova, Scott Kitchen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Supplementary information

Supplementary Video 1

NeoThy surgery: nicking the kidney capsule. A shallow nick is made to cut through the kidney capsule.

Supplementary Video 2

Creating a tissue pocket in the kidney capsule. A tissue pocket is made by gently grabbing the nicked opening with a fine forceps and using the end of a bent blunt end forceps to create a pocket approximately 1 cm × 1 cm in size. Kidney is moistened with sterile PBS swab to prevent it from drying.

Supplementary Video 3

Insertion of neonatal thymus fragments under the kidney capsule. By gently grabbing and slightly lifting up the nicked opening with a fine forceps, the bent blunt end forceps is used to grab the edge of the thymus fragment, inserting into the pocket. The rounded back side of the bent blunt forceps is used to push the fragment to the end of the kidney, away from the nick. This procedure is repeated with a second thymus fragment, taking care not to tear the kidney capsule.

Supplementary Data 1

HLA-typing melt curve data. This file contains an example of HLA typing data, which can be used as practice for generating a report before working on actual samples. It contains an ‘ambiguous rare’ call that can be manually selected by the user, as this may be helpful practice of an issue that can arise with these samples.

Supplementary Data 2

HLA detailed report. This file contains an example of the final LinkSeq HLA-typing report generated from the data in Supplementary Data 1.

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Del Rio, N.M., Huang, L., Murphy, L. et al. Generation of the NeoThy mouse model for human immune system studies. Lab Anim 52, 149–168 (2023).

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