@article{schaaf_polkoff_carter_stewart_sheahan_freund_ginzel_snyder_roper_piedrahita_et al._2023, title={A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease}, volume={37}, ISSN={["1530-6860"]}, DOI={10.1096/fj.202300223R}, abstractNote={Abstract Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein‐Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence‐activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5‐H2B‐GFP and wild‐type pigs. Ileum and colon LGR5‐H2B‐GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli ( APC ) mutation was induced by CRISPR/Cas9 editing in porcine LGR5‐H2B‐GFP colonoids. Crypt‐base, green fluorescent protein (GFP) expressing cells co‐localized with ISC biomarkers. LGR5‐H2B‐GFP hi cells had significantly higher LGR5 expression ( p < .01) and enteroid forming efficiency ( p < .0001) compared with LGR5‐H2B‐GFP med/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ , and SOX9 expression was identified between human and LGR5‐H2B‐GFP pig crypt‐base cells. LGR5‐H2B‐GFP/ APC null colonoids had cystic growth in WNT/R‐spondin‐depleted media and significantly upregulated WNT/β‐catenin target gene expression ( p < .05). LGR5 + ISCs are reproducibly isolated in LGR5‐H2B‐GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt‐base FISH, underscore the significance of this novel LGR5‐H2B‐GFP pig to translational ISC research.}, number={6}, journal={FASEB JOURNAL}, author={Schaaf, Cecilia R. and Polkoff, Kathryn M. and Carter, Amber and Stewart, Amy S. and Sheahan, Breanna and Freund, John and Ginzel, Joshua and Snyder, Joshua C. and Roper, Jatin and Piedrahita, Jorge A. and et al.}, year={2023}, month={Jun} }
 @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 −/− IL2RG y /− 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{detwiler_polkoff_gaffney_freytes_piedrahita_2022, title={Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in an In Vitro Hydrogel Model}, ISSN={["1937-335X"]}, DOI={10.1089/ten.tea.2021.0217}, abstractNote={Current cellular hydrogel-based skin grafts composed of human dermal fibroblasts and a hydrogel scaffold tend to minimize contraction of full-thickness skin wounds and support skin regeneration. However, there has been no comparison between the sources of the dermal fibroblast used. Products using human adult or neonatal foreskin dermal fibroblasts are often expanded in vitro and used after multiple passages without a clear understanding of the effects of this initial production step on the quality and reproducibility of the cellular behavior. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may influence cellular properties and contractile behavior in a fibroblast-populated collagen matrix. Using porcine skin as a model based on its similarity to human skin in structure and wound healing properties, we isolated porcine dermal fibroblasts of three different donor ages for use in a 2D proliferation assay and in a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1 in vitro. However, after five passages in culture, there was no difference in contractility between ages. These results show how cellular responses in a hydrogel scaffold differ based on donor age and time in culture in vitro, and suggest that consistency in the cellular component of bioengineered skin products could be beneficial in the biomanufacturing of consistent, reliable skin grafts and graft in vivo models. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding. Impact statement Little is known about the impact of donor cell age and time in culture on the contraction of cellular, hydrogel-based skin grafts. These results show how cellular phenotypes of porcine fibroblasts differ based on donor age and time in culture. This information is beneficial when addressing important inconsistencies in biomanufacturing of bioengineered skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. The information from this study can be extrapolated to future in vivo studies using human dermal fibroblasts in an in vivo model to help determine the best donor age and time in culture for optimal wound healing outcomes or more reproducible in vitro testing constructs.}, journal={TISSUE ENGINEERING PART A}, author={Detwiler, Amber and Polkoff, Kathryn and Gaffney, Lewis and Freytes, Donald O. and Piedrahita, Jorge A.}, year={2022}, month={Aug} }
 @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 Hair follicle stem cells are key for driving growth and homeostasis of the hair follicle niche, have remarkable regenerative capacity throughout hair cycling, and display fate plasticity during cutaneous wound healing. Due to the need for a transgenic reporter, essentially all observations related to LGR5-expressing hair follicle stem cells have been generated using transgenic mice, which have significant differences in anatomy and physiology from the human. Using a transgenic pig model, a widely accepted model for human skin and human skin repair, we demonstrate that LGR5 is a marker of hair follicle stem cells across species in homeostasis and development. We also report the strong similarities and important differences in expression patterns, gene expression profiles, and developmental processes between species. This information is important for understanding the fundamental differences and similarities across species, and ultimately improving human hair follicle regeneration, cutaneous wound healing, and skin cancer treatment.}, 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{chansoria_asif_polkoff_chung_piedrahita_shirwaiker_2021, title={Characterizing the Effects of Synergistic Thermal and Photo-Cross-Linking during Biofabrication on the Structural and Functional Properties of Gelatin Methacryloyl (GeIMA) Hydrogels}, volume={7}, ISSN={["2373-9878"]}, DOI={10.1021/acsbiomaterials.1c00635}, abstractNote={Gelatin methacryloyl (GelMA) hydrogels have emerged as promising and versatile biomaterial matrices with applications spanning drug delivery, disease modeling, and tissue engineering and regenerative medicine. GelMA exhibits reversible thermal cross-linking at temperatures below 37 °C due to the entanglement of constitutive polymeric chains, and subsequent ultraviolet (UV) photo-cross-linking can covalently bind neighboring chains to create irreversibly cross-linked hydrogels. However, how these cross-linking modalities interact and can be modulated during biofabrication to control the structural and functional characteristics of this versatile biomaterial is not well explored yet. Accordingly, this work characterizes the effects of synergistic thermal and photo-cross-linking as a function of GelMA solution temperature and UV photo-cross-linking duration during biofabrication on the hydrogels' stiffness, microstructure, proteolytic degradation, and responses of NIH 3T3 and human adipose-derived stem cells (hASC). Smaller pore size, lower degradation rate, and increased stiffness are reported in hydrogels processed at lower temperature or prolonged UV exposure. In hydrogels with low stiffness, the cells were found to shear the matrix and cluster into microspheroids, while poor cell attachment was noted in high stiffness hydrogels. In hydrogels with moderate stiffness, ones processed at lower temperature demonstrated better shape fidelity and cell proliferation over time. Analysis of gene expression of hASC encapsulated within the hydrogels showed that, while the GelMA matrix assisted in maintenance of stem cell phenotype (CD44), a higher matrix stiffness resulted in higher pro-inflammatory marker (ICAM1) and markers for cell-matrix interaction (ITGA1 and ITGA10). Analysis of constructs with ultrasonically patterned hASC showed that hydrogels processed at higher temperature possessed lower structural fidelity but resulted in more cell elongation and greater anisotropy over time. These findings demonstrate the significant impact of GelMA material formulation and processing conditions on the structural and functional properties of the hydrogels. The understanding of these material-process-structure-function interactions is critical toward optimizing the functional properties of GelMA hydrogels for different targeted applications.}, number={11}, journal={ACS BIOMATERIALS SCIENCE & ENGINEERING}, author={Chansoria, Parth and Asif, Suleman and Polkoff, Kathryn and Chung, Jaewook and Piedrahita, Jorge A. and Shirwaiker, Rohan A.}, year={2021}, month={Nov}, pages={5175–5188} }
 @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} }