Jorge A. Piedrahita

Cells expressing leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) play a pivotal role in homeostasis, repair, and regeneration in multiple organs, including skin and gastrointestinal tract, but little is known about their role in the lung. Findings from mice, a widely used animal model, suggest that lung LGR5 expression differs from that of humans. In this work, using a new transgenic pig model, we identify two main populations of LGR5+ cells in the lung that are conserved in human but not mouse lungs. Using RNA sequencing, three-dimensional imaging, and organoid models, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of SOX9+/ETV5+/SFTPC+ progenitor lung tip cells. In contrast, epithelial LGR5 expression is absent from postnatal lung but is reactivated in bronchioalveolar organoids derived from basal airway cells. We also describe a separate population of mesenchymal LGR5+ cells that surrounds developing and mature airways, lies adjacent to airway basal cells, and is closely associated with nerve fibers. Transcriptionally, mesenchymal LGR5+ cells include a subset of peribronchial fibroblasts that express unique patterns of SHH, FGF, WNT, and TGF-β signaling pathway genes. These results support distinct roles for LGR5+ cells in the lung and describe a physiologically relevant animal model for further studies on the function of these cells in repair and regeneration.

Angelman syndrome is a neurodevelopmental disorder characterized by severe motor and cognitive deficits. It is caused by the loss of the maternally inherited allele of the imprinted ubiquitin-protein ligase E3A ( UBE3A ) gene. Rodent models of Angelman syndrome do not fully recapitulate all the symptoms associated with the condition and are limited as a preclinical model for therapeutic development. Here, we show that pigs ( Sus scrofa ) with a maternally inherited deletion of UBE3A ( UBE3A ^−/+ ) have altered postnatal behaviors, impaired vocalizations, reduced brain growth, motor incoordination, and ataxia. Neonatal UBE3A ^−/+ pigs exhibited several symptoms observed in infants with Angelman syndrome, including hypotonia, suckling deficits, and failure to thrive. Collectively, these findings are consistent with the pathophysiology and developmental trajectory observed in individuals with Angelman syndrome. We anticipate that this pig model will advance our understanding of the pathophysiology of Angelman syndrome and be used as a preclinical large animal model for therapeutic development.

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. The model was designed to induce the expression of H2B-GFP under the regulatory control of the LGR5 promoter via CRISPR/Cas9-mediated gene knock-in. Notably, advancements in stem cell research have identified distinct subpopulations of LGR5-expressing cells within adult human, mouse, and pig tissues. LGR5, a leucine-rich repeat-containing G protein-coupled receptor, enhances WNT signaling and these LGR5⁺ subpopulations demonstrate varied roles and anatomical distributions, underscoring the necessity for suitable translational models. This transgenic pig model facilitates the tracking of LGR5-expressing cells and has provided valuable insights into the roles of these cells across different tissues and species. For instance, in pulmonary tissue, Lgr5⁺ cells in mice are predominantly located in alveolar compartments, driving alveolar differentiation of epithelial progenitors via Wnt pathway activation. In contrast, in pigs and humans, these cells are situated in a unique sub-basal position adjacent to the airway epithelium. In fetal stages a pattern of LGR5 expression during lung bud tip formation is evident in humans and pigs but is lacking in mice. Species differences with respect to LGR5 expression have also been observed in the skin, intestines, and cochlea further reinforcing the need for careful selection of appropriate translational animal models. This paper discusses the potential utility of the LGR5⁺ pig model in exploring the role of LGR5⁺ cells in tissue development and regeneration with the goal of translating these findings into human and animal clinical applications.

Developing a new drug can cost as much as a billion dollars, mostly due to the very large number of candidate drugs that fail before they get to the patient. Thus, finding ways to identify those “bad” drugs early can have a huge impact in reducing the cost of drugs and that in turn will drastically impact global health. Drs. Piedrahita and Pierce discuss how the use of mini-organs -- organoids made from stem cells -- can play a role in solving this wicked problem. They address some of the limitations of organoids and what is being done to overcome those limitations.

A network of co-hepato/pancreatic stem/progenitors exists in pigs and humans in Brunner’s Glands in the submucosa of the duodenum, in peribiliary glands (PBGs) of intrahepatic and extrahepatic biliary trees, and in pancreatic duct glands (PDGs) of intrapancreatic biliary trees, collectively supporting hepatic and pancreatic regeneration postnatally. The network is found in humans postnatally throughout life and, so far, has been demonstrated in pigs postnatally at least through to young adulthood. These stem/progenitors in vivo in pigs are in highest numbers in Brunner’s Glands and in PDGs nearest the duodenum, and in humans are in Brunner’s Glands and in PBGs in the hepato/pancreatic common duct, a duct missing postnatally in pigs. Elsewhere in PDGs in pigs and in all PDGs in humans are only committed unipotent or bipotent progenitors. Stem/progenitors have genetic signatures in liver/pancreas-related RNA-seq data based on correlation, hierarchical clustering, differential gene expression and principal component analyses (PCA). Gene expression includes representative traits of pluripotency genes (SOX2, OCT4), endodermal transcription factors (e.g. SOX9, SOX17, PDX1), other stem cell traits (e.g. NCAM, CD44, sodium iodide symporter or NIS), and proliferation biomarkers (Ki67). Hepato/pancreatic multipotentiality was demonstrated by the stem/progenitors’ responses under distinct ex vivo conditions or in vivo when patch grafted as organoids onto the liver versus the pancreas. Therefore, pigs are logical hosts for translational/preclinical studies for cell therapies with these stem/progenitors for hepatic and pancreatic dysfunctions.

Cells expressing LGR5 play a pivotal role in homeostasis, repair, and regeneration in multiple organs including skin and gastrointestinal tract, yet little is known about their role in the lung. Findings from mice, a widely used animal model, suggest that lung LGR5 expression differs from that of humans. In this work, using a new transgenic pig model, we identify two main populations of LGR5 ⁺ cells in the lung that are conserved in human, but not mouse lungs. Using RNA sequencing, 3D imaging and organoid models, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of SOX9 ⁺ /ETV ⁺ /SFTPC ⁺ progenitor lung tip cells. In contrast, epithelial LGR5 expression is absent from postnatal lung, but is reactivated in bronchioalveolar organoids derived from basal airway cells. We also describe a separate population of mesenchymal LGR5 ⁺ cells that surrounds developing and mature airways, lies adjacent to airway basal cells, and is closely associated with nerve fibers. Transcriptionally, mesenchymal LGR5 ⁺ cells include a subset of peribronchial fibroblasts (PBF) that express unique patterns of SHH, FGF, WNT and TGF- β signaling pathway genes. These results support distinct roles for LGR5 ⁺ cells in the lung and describe a physiologically relevant animal model for further studies on the function of these cells in repair and regeneration.

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.

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.