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.

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.

Stem cells play a pivotal role in lung homeostasis, repair, and regeneration, and yet the mechanisms underlying these processes are unknown. Furthermore, species-specific differences make certain findings from mice, a widely used animal model, difficult to translate into humans. In this work, we address these limitations by using a transgenic pig model and identify two populations of LGR5+ cells in the lung. Interestingly, we found similar populations in human lungs but not in mice. Using RNA sequencing, 3D imaging, organoid models, and differentiation assays, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of developing lung bud tips, co-expresses bud tip markers SOX9 and SFTPC. While epithelial LGR5 expression is absent from postnatal lung, it is reactivated in bronchioalveolar organoids derived from basal airway cells. A separate population of LGR5+ cells is mesenchymal, surrounds developing and mature airways, lies adjacent to airway basal cells, is closely associated with nerve fibers, and acts as a multipotent progenitor cell capable of supporting the airway basal cell niche. Transcriptionally, mesenchymal LGR5+ cells are analogous to stromal stem cell populations and express unique patterns of WNT and TGFbeta signaling pathway genes. These results point to two roles for LGR5 in orchestrating stem and progenitor cell dynamics and provide a physiologically relevant model for further studies on the role of these populations in repair and regeneration.

Hair follicle stem cells are key for driving growth and homeostasis of the hair follicle niche and 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+ 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.