ORIGINAL RESEARCH article
Front. Immunol.
Sec. Alloimmunity and Transplantation
Midgestational injection of highly expanded human CD34+ cells increases lineages of human immune cells and supports thymic development in RAG2-/-IL2RG-/Y SCID pigs
Ahlea Forster 1
Amanda Kress 2
Matti Kiupel 3
Joan E. Cunnick 1
Dennis Webster 4
Jarryd Campbell 4
Adrienne Watson 4
Ohad Gafni 4
Daniel F. Carlson 4
Branden Scott Moriarity 5
Beau R. Webber 5
Brett Napiwocki 5
Lance Daharsh 1
Jason Ross 1
Mary Sauer 2
Christopher K Tuggle 1
1. Iowa State University of Science and Technology, Ames, United States
2. Laboratory Animal Resources, Iowa State University, Ames, United States
3. Michigan State University Veterinary Diagnostic Laboratory, Lansing, United States
4. Recombinetics Inc, Eagan, United States
5. University of Minnesota Twin Cities Department of Pediatrics, Minneapolis, United States
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Abstract
Severe combined immunodeficiency (SCID) pigs have become a promising large animal model for biomedical research, offering significant advantages over traditional mouse models due to their anatomical, physiological, and genetic similarities to humans. Humanized SCID pig models can potentially improve preclinical research in areas such as cancer immunotherapies, stem cell therapies, and transplantation methods, yet often lack significant lymphocyte development, including evidence of B cell and myeloid cell development. This work aims to increase the extent of humanization of the SCID pig. CRISPR guide RNAs were successfully developed for the RAG2 and IL2RG genes, and a double-knockout cell line (RAG2-/-IL2RG-/Y, RG) was established. Somatic cell nuclear transfer (SCNT) was then used to create cloned SCID fetuses, which were injected intraperitoneally with in vitro expanded human CD34+ umbilical cord cells at day 41-42 of gestation. Human leukocytes, including T, B, NK, and myeloid cell types, were detected in peripheral blood, spleen, bone marrow and within the thymus of neonatal animals using flow cytometry. Six of the twelve pigs injected had >5% human cells within the CD45+ cell thymic population. Histology of thymus tissues from multiple pigs showed substantial development of the cortex and medulla, which is absent in non-injected RG neonates. This work demonstrates an improvement in the spectrum of xenogenic This is a provisional file, not the final typeset article immune cell lineages developed using an RG line injected with highly expanded CD34+ cells, yet functional analysis of these cell types is needed for further establishment of an in utero humanized SCID pig model.
Summary
Keywords
Biomedical model development3, Humanization2, SCID-severe combined immunodeficiency1, swine4, Translational biomedicine5
Received
21 November 2025
Accepted
20 February 2026
Copyright
© 2026 Forster, Kress, Kiupel, Cunnick, Webster, Campbell, Watson, Gafni, Carlson, Moriarity, Webber, Napiwocki, Daharsh, Ross, Sauer and Tuggle. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Ahlea Forster; Christopher K Tuggle
Disclaimer
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