Midgestational injection of highly expanded human CD34+ cells increases lineages of human immune cells and supports thymic development in RAG2-/-IL2RG-/Y SCID pigs

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 <i>in vitro</i> 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 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 <i>in utero</i> humanized SCID pig model.