@article{sper_proctor_lascina_guo_polkoff_kaeser_simpson_borst_gleason_zhang_et al._2022, title={Allogeneic and xenogeneic lymphoid reconstitution in a RAG2(-/-)IL2RG(y/-) severe combined immunodeficient pig: A preclinical model for intrauterine hematopoietic transplantation}, volume={9}, ISSN={["2297-1769"]}, DOI={10.3389/fvets.2022.965316}, abstractNote={Mice with severe combined immunodeficiency are commonly used as hosts of human cells. Size, longevity, and physiology, however, limit the extent to which immunodeficient mice can model human systems. To address these limitations, we generated RAG2−/−IL2RGy/− immunodeficient pigs and demonstrate successful engraftment of SLA mismatched allogeneic D42 fetal liver cells, tagged with pH2B-eGFP, and human CD34+ hematopoietic stem cells after in utero cell transplantation. Following intrauterine injection at day 42–45 of gestation, fetuses were allowed to gestate to term and analyzed postnatally for the presence of pig (allogeneic) and human (xenogeneic) B cells, T-cells and NK cells in peripheral blood and other lymphoid tissues. Engraftment of allogeneic hematopoietic cells was detected based on co-expression of pH2B-eGFP and various markers of differentiation. Analysis of spleen revealed robust generation and engraftment of pH2B-eGFP mature B cells (and IgH recombination) and mature T-cells (and TCR-β recombination), T helper (CD3+CD4+) and T cytotoxic (CD3+CD8+) cells. The thymus revealed engraftment of pH2B-eGFP double negative precursors (CD4−CD8−) as well as double positive (CD4+, CD8+) precursors and single positive T-cells. After intrauterine administration of human CD34+ hematopoietic stem cells, analysis of peripheral blood and lymphoid tissues revealed the presence of human T-cells (CD3+CD4+ and CD3+CD8+) but no detectable B cells or NK cells. The frequency of human CD45+ cells in the circulation decreased rapidly and were undetectable within 2 weeks of age. The frequency of human CD45+ cells in the spleen also decreased rapidly, becoming undetectable at 3 weeks. In contrast, human CD45+CD3+T-cells comprised >70% of cells in the pig thymus at birth and persisted at the same frequency at 3 weeks. Most human CD3+ cells in the pig's thymus expressed CD4 or CD8, but few cells were double positive (CD4+ CD8+). In addition, human CD3+ cells in the pig thymus contained human T-cell excision circles (TREC), suggesting de novo development. Our data shows that the pig thymus provides a microenvironment conducive to engraftment, survival and development of human T-cells and provide evidence that the developing T-cell compartment can be populated to a significant extent by human cells in large animals.}, journal={FRONTIERS IN VETERINARY SCIENCE}, author={Sper, Renan B. and Proctor, Jessica and Lascina, Odessa and Guo, Ling and Polkoff, Kathryn and Kaeser, Tobias and Simpson, Sean and Borst, Luke and Gleason, Katherine and Zhang, Xia and et al.}, year={2022}, month={Oct} }
 @article{polkoff_gupta_green_murphy_chung_gleason_simpson_walker_collins_piedrahita_2022, title={LGR5 is a conserved marker of hair follicle stem cells in multiple species and is present early and throughout follicle morphogenesis}, volume={12}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-022-13056-w}, abstractNote={Abstract}, number={1}, journal={SCIENTIFIC REPORTS}, author={Polkoff, Kathryn M. and Gupta, Nithin K. and Green, Adrian J. and Murphy, Yanet and Chung, Jaewook and Gleason, Katherine L. and Simpson, Sean G. and Walker, Derek M. and Collins, Bruce and Piedrahita, Jorge A.}, year={2022}, month={Jun} }
 @article{zhang_lanzoni_hani_overi_cardinale_simpson_pitman_allen_yi_wang_et al._2021, title={Patch grafting, strategies for transplantation of organoids into solid organs such as liver}, volume={277}, ISSN={["1878-5905"]}, DOI={10.1016/j.biomaterials.2021.121067}, abstractNote={Epithelial cell therapies have been at an impasse because of inefficient methods of transplantation to solid organs. Patch grafting strategies were established enabling transplantation of ≥107th organoids/patch of porcine GFP+ biliary tree stem/progenitors into livers of wild type hosts. Grafts consisted of organoids embedded in soft (~100 Pa) hyaluronan hydrogels, both prepared in serum-free Kubota's Medium; placed against target sites; covered with a silk backing impregnated with more rigid hyaluronan hydrogels (~700 Pa); and use of the backing to tether grafts with sutures or glue to target sites. Hyaluronan coatings (~200-300 Pa) onto the serosal surface of the graft served to minimize adhesions with neighboring organs. The organ's clearance of hyaluronans enabled restoration of tissue-specific paracrine and systemic signaling, resulting in return of normal hepatic histology, with donor parenchymal cells uniformly integrated amidst host cells and that had differentiated to mature hepatocytes and cholangiocytes. Grafts containing donor mature hepatocytes, partnered with endothelia, and in the same graft biomaterials as for stem/progenitor organoids, did not engraft. Engraftment occurred if porcine liver-derived mesenchymal stem cells (MSCs) were co-transplanted with donor mature cells. RNA-seq analyses revealed that engraftment correlated with expression of matrix-metalloproteinases (MMPs), especially secreted isoforms that were found expressed strongly by organoids, less so by MSCs, and minimally, if at all, by adult cells. Engraftment with patch grafting strategies occurred without evidence of emboli or ectopic cell distribution. It was successful with stem/progenitor organoids or with cells with a source(s) of secreted MMP isoforms and offers significant potential for enabling cell therapies for solid organs.}, journal={BIOMATERIALS}, author={Zhang, Wencheng and Lanzoni, Giacomo and Hani, Homayoun and Overi, Diletta and Cardinale, Vincenzo and Simpson, Sean and Pitman, Wendy and Allen, Amanda and Yi, Xianwen and Wang, Xicheng and et al.}, year={2021}, month={Oct} }
 @article{polkoff_chung_simpson_gleason_piedrahita_2020, title={In Vitro Validation of Transgene Expression in Gene-Edited Pias Using CRISPR Transcriptional Activators}, volume={3}, ISSN={["2573-1602"]}, DOI={10.1089/crispr.2020.0037}, abstractNote={The use of CRISPR-Cas and RNA-guided endonucleases has drastically changed research strategies for understanding and exploiting gene function, particularly for the generation of gene-edited animal models. This has resulted in an explosion in the number of gene-edited species, including highly biomedically relevant pig models. However, even with error-free DNA insertion or deletion, edited genes are occasionally not expressed and/or translated as expected. Therefore, there is a need to validate the expression outcomes gene modifications in vitro before investing in the costly generation of a gene-edited animal. Unfortunately, many gene targets are tissue specific and/or not expressed in cultured primary cells, making validation difficult without generating an animal. In this study, using pigs as a proof of concept, we show that CRISPR-dCas9 transcriptional activators can be used to validate functional transgene insertion in nonexpressing easily cultured cells such as fibroblasts. This is a tool that can be used across disciplines and animal species to save time and resources by verifying expected outcomes of gene edits before generating live animals.}, number={5}, journal={CRISPR JOURNAL}, author={Polkoff, Kathryn M. and Chung, Jaewook and Simpson, Sean G. and Gleason, Katherine and Piedrahita, Jorge A.}, year={2020}, month={Oct}, pages={409–418} }
 @article{chung_zhang_collins_sper_gleason_simpson_koh_sommer_flowers_petters_et al._2018, title={High mobility group A2 (HMGA2) deficiency in pigs leads to dwarfism, abnormal fetal resource allocation, and cryptorchidism}, volume={115}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1721630115}, DOI={10.1073/pnas.1721630115}, abstractNote={Significance}, number={21}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Chung, Jaewook and Zhang, Xia and Collins, Bruce and Sper, Renan B. and Gleason, Katherine and Simpson, Sean and Koh, Sehwon and Sommer, Jeffrey and Flowers, William L. and Petters, Robert M. and et al.}, year={2018}, month={May}, pages={5420–5425} }
 @article{jeong_nelson_niedziela_dickey_2016, title={Effect of Plant Species, Fertilizer Acidity/Basicity, and Fertilizer Concentration on pH of Soilless Root Substrate}, volume={51}, ISSN={["2327-9834"]}, DOI={10.21273/hortsci11237-16}, abstractNote={The objective of this study was to determine how plant species, fertilizer potential acidity/basicity rating (PABR), and fertilizer concentration affect root substrate pH. Three experiments were conducted. In the first experiment, 13 herbaceous species were grown in a root substrate of three sphagnum peatmoss: one perlite (v/v) with deionized water and a neutral fertilizer (NF) with a PABR of 0 for 78 days to determine species relationships to substrate pH. The decrease in substrate pH ranged from 0.14 to 2.45 units, depending on species. In the second experiment, four of the 13 species from the previous trial representing the range of pH suppression were grown under similar growth conditions as the first experiment for 70 days. Substrate pH was lowered in the range of 0.47 to 2.72 units. In the third experiment, three fertilizers with PABRs of 150 kg·t−1 CaCO3 equivalent alkalinity, 0 neutral, and 193 kg·t−1 CaCO3 equivalent acidity were applied in a factorial design at 100 and 200 mg·L−1 N at each irrigation to kalanchoe (the species with the greatest pH suppression from the previous experiments) for 56 days. When applied at the lower fertilizer rate (100 mg·L−1 N), the PABRs resulted in the final substrate pH levels of 4.68, 5.60, and 6.11 for the acidic fertilizer (AF), NF, and basic fertilizer (BF), respectively. At the high fertilizer rate (200 mg·L−1 N), substrate pH declined continuously to 3.97, 4.03, and 4.92 for the AF, NF, and BF, respectively. Expression of PABR depended on the balance between the abiotic (chemical) effect of the fertilizers vs. the biotic (physiological) effects of the fertilizers on microbes and plants. The PABR was best expressed when the fertilizer supply was just adequate or lower indicating a closer connection to the biotic effect.}, number={12}, journal={HORTSCIENCE}, author={Jeong, Ka Yeon and Nelson, Paul V. and Niedziela, Carl E., Jr. and Dickey, David A.}, year={2016}, month={Dec}, pages={1596–1601} }
 @article{sommer_chavali_simpson_ayyagari_petters_2012, title={Cloning, characterization, and expression analysis of the pig (Sus scrofa) C1q tumor necrosis factor-related protein-5 gene}, volume={18}, number={12-14}, journal={Molecular Vision}, author={Sommer, J. R. and Chavali, V. R. M. and Simpson, S. G. and Ayyagari, R. and Petters, R. M.}, year={2012}, pages={92–102} }
 @article{sommer_jackson_simpson_collins_piedrahita_petters_2012, title={Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies}, volume={21}, ISSN={["0962-8819"]}, DOI={10.1007/s11248-011-9542-6}, abstractNote={The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.}, number={2}, journal={TRANSGENIC RESEARCH}, author={Sommer, Jeffrey R. and Jackson, Lauren R. and Simpson, Sean G. and Collins, Edwin B. and Piedrahita, Jorge A. and Petters, Robert M.}, year={2012}, month={Apr}, pages={383–392} }