@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={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} }
 @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={Expression of HMGA2 is strongly associated with body size and growth in mice and humans. In mice, inactivation of one or both alleles of Hmga2 results in body-size reductions of 20% and 60%, respectively. In humans, microdeletions involving the HMGA2 locus result in short stature, suggesting the function of the HMGA2 protein is conserved among mammals. To test this hypothesis, we generated HMGA2-deficient pigs via gene editing and somatic cell nuclear transfer (SCNT). Examination of growth parameters revealed that HMGA2-/+ male and female pigs were on average 20% lighter and smaller than HMGA2+/+ matched controls (P < 0.05). HMGA2-/- boars showed significant size reduction ranging from 35 to 85% of controls depending on age (P < 0.05), and organ weights were also affected (P < 0.05). HMGA2-/+ gilts and boars exhibited normal reproductive development and fertility, while HMGA2-/- boars were sterile due to undescended testes (cryptorchidism). Crossbreeding HMGA2-/+ boars and gilts produced litters lacking the HMGA2-/- genotype. However, analysis of day (D) D40 and D78 pregnancies indicated that HMGA2-/- fetuses were present at the expected Mendelian ratio, but placental abnormalities were seen in the D78 HMGA2-/- concepti. Additionally, HMGA2-/- embryos generated by gene editing and SCNT produced multiple pregnancies and viable offspring, indicating that lack of HMGA2 is not lethal per se. Overall, our results show that the effect of HMGA2 with respect to growth regulation is highly conserved among mammals and opens up the possibility of regulating body and organ size in a variety of mammalian species including food and companion animals.}, 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{chung_tsai_james_thames_shytle_piedrahita_2012, title={Lack of genomic imprinting of DNA primase, polypeptide 2 (PRIM2) in human term placenta and white blood cells}, volume={7}, ISSN={["1559-2294"]}, DOI={10.4161/epi.19777}, abstractNote={PRIM2, encoding a subunit of primase involved in DNA replication and transcription, is expressed in the placenta and is crucial for mammalian development and growth. Its role in placental function is not well understood. Recently, PRIM2 was reported as imprinted in human white blood cells (WBC). We report here our failure to confirm imprinting of the PRIM2 locus in human placenta or WBC. The discordance between our results and those of others are likely due to an incorrectly annotated PRIM2 pseudogene found in the human genome database.}, number={5}, journal={EPIGENETICS}, author={Chung, Jaewook and Tsai, Shengdar and James, Andra H. and Thames, Betty H. and Shytle, Stephanie and Piedrahita, Jorge A.}, year={2012}, month={May}, pages={429–431} }