A gene edited pig model for studying LGR5 stem cells: implications for future applications in tissue regeneration and biomedical research.

Recent advancements in genome editing techniques, notably CRISPR-Cas9 and TALENs, have marked a transformative era in biomedical research, significantly enhancing our understanding of disease mechanisms and helping develop novel therapies. These technologies have been instrumental in creating precise animal models for use in stem cell research and regenerative medicine. For instance, we have developed a transgenic pig model to enable the investigation of LGR5-expressing cells.

A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease.

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.

Uterine histotroph and conceptus development: III. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via AKT-TSC2-MTOR cell signaling pathway.

Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr2) isolated from Day-12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via activation of mechanistic target of rapamycin (MTOR) cell signaling pathway in pTr2 cells. Porcine ADM at 10 M stimulated (P < 0.

Inhalable exosomes outperform liposomes as mRNA and protein drug carriers to the lung.

Respiratory diseases are among the leading causes of morbidity and mortality worldwide, coupled with the ongoing coronavirus disease 2019 (COVID-19) pandemic. mRNA lipid nanoparticle (LNP) vaccines have been developed, but their intramuscular delivery limits pulmonary bioavailability. Inhalation of nanoparticle therapeutics offers localized drug delivery that minimizes off targeted adverse effects and has greater patient compliance.

Allogeneic and xenogeneic lymphoid reconstitution in a severe combined immunodeficient pig: A preclinical model for intrauterine hematopoietic transplantation.

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 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 cell transplantation.

Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing.

Recombinant adeno-associated viral (AAV) vectors are a promising gene delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Effective clinical translation is confounded, at least in part, by differences in AAV biology across animal species. Here, we tackle this challenge by sequentially evolving AAV capsid libraries in mice, pigs and macaques.

Patch grafting of organoids of stem/progenitors into solid organs can correct genetic-based disease states.

Patch grafting, a novel strategy for transplantation of stem/progenitor organoids into porcine livers, has been found successful also for organoid transplantation into other normal or diseased solid organs in pigs and mice. Each organoid contained ∼100 cells comprised of biliary tree stem cells (BTSCs), co-hepato/pancreatic stem/progenitors, and partnered with early lineage stage mesenchymal cells (ELSMCs), angioblasts and precursors to endothelia and stellate cells. Patch grafting enabled transplantation into livers or pancreases of ≥10 (pigs) or ≥10 (mice) organoids/patch.

The use of autologous skeletal muscle progenitor cells for adjunctive treatment of presumptive urethral sphincter mechanism incompetence in female dogs.

Background: Urethral sphincter mechanism incompetence (USMI) is a common problem in female dogs, but some dogs fail to achieve continence with standard treatment. Urethral submucosal injection of autologous skeletal muscle progenitor cells (skMPCs) previously has been shown to restore urethral function in a canine model of USMI.

Hypothesis/objective: To determine if urethral submucosal injection of skMPC alters continence in dogs with USMI that had previously failed standard medical management.

Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in an Hydrogel Model.

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 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.

Ontogeny of cellular organization and LGR5 expression in porcine cochlea revealed using tissue clearing and 3D imaging.

Over 11% of the world's population experience hearing loss. Although there are promising studies to restore hearing in rodent models, the size, ontogeny, genetics, and frequency range of hearing of most rodents' cochlea do not match that of humans. The porcine cochlea can bridge this gap as it shares many anatomical, physiological, and genetic similarities with its human counterpart.

LGR5 is a conserved marker of hair follicle stem cells in multiple species and is present early and throughout follicle morphogenesis.

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.

Multiscale Anisotropic Tissue Biofabrication via Bulk Acoustic Patterning of Cells and Functional Additives in Hybrid Bioinks.

Recapitulation of the microstructural organization of cellular and extracellular components found in natural tissues is an important but challenging feat for tissue engineering, which demands innovation across both process and material fronts. In this work, a highly versatile ultrasound-assisted biofabrication (UAB) approach is demonstrated that utilizes radiation forces generated by superimposing ultrasonic bulk acoustic waves to rapidly organize arrays of cells and other biomaterial additives within single and multilayered hydrogel constructs. UAB is used in conjunction with a novel hybrid bioink system, comprising of cartilage-forming cells (human adipose-derived stem cells or chondrocytes) and additives to promote cell adhesion (collagen microaggregates or polycaprolactone microfibers) encapsulated within gelatin methacryloyl (GelMA) hydrogels, to fabricate cartilaginous tissue constructs featuring bulk anisotropy.

Age- and Sex-Specific Joint Biomechanics in Response to Partial and Complete Anterior Cruciate Ligament Injury in the Porcine Model.

Context: Pediatric anterior cruciate ligament (ACL) injury rates are increasing and are highest in female adolescents. Complete ACL tears are typically surgically reconstructed, but few guidelines and very limited data exist regarding the need for surgical reconstruction or rehabilitation for partial ACL tears in skeletally immature patients.

Objective: To evaluate the effects of partial (anteromedial bundle) and complete ACL transection on joint laxity and tissue forces under anterior and rotational loads in male and female stifle joints throughout skeletal growth in the porcine model.

Sex-specific biomechanics and morphology of the anterior cruciate ligament during skeletal growth in a porcine model.

Pediatric anterior cruciate ligament (ACL) injuries are on the rise, and females experience higher ACL injury risk than males during adolescence. Studies in skeletally immature patients indicate differences in ACL size and joint laxity between males and females after the onset of adolescence. However, functional data regarding the ACL and its anteromedial and posterolateral bundles in the pediatric population remain rare.

Patch grafting, strategies for transplantation of organoids into solid organs such as liver.

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 ≥10 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.

Three-dimensional imaging of intact porcine cochlea using tissue clearing and custom-built light-sheet microscopy.

Hearing loss is a prevalent disorder that affects people of all ages. On top of the existing hearing aids and cochlear implants, there is a growing effort to regenerate functional tissues and restore hearing. However, studying and evaluating these regenerative medicine approaches in a big animal model (e.

Validation of Transgene Expression in Gene-Edited Pigs Using CRISPR Transcriptional Activators.

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.

Fatal neurocryptococcosis in a Colombian underage patient.

Cryptococcosis is a life-threatening mycosis reported mainly in human adults with low frequency in children. High mortality rates may occur in cases with late diagnosis therefore, a timely suspicion of this pathology is important. Cryptococcus gattii is the less prevalent species complex predominantly isolated from apparently normal hosts.

Joint laxity varies in response to partial and complete anterior cruciate ligament injuries throughout skeletal growth.

Anterior cruciate ligament (ACL) injuries are increasingly common in the skeletally immature population. As such there is a need to increase our understanding of the biomechanical function of the joint following partial and complete ACL injury during skeletal growth. In this work, we aimed to assess changes in knee kinematics and loading of the remaining soft tissues following both partial and complete ACL injury in a porcine model.