Quiescence, the reversible exit from the proliferative cell cycle, is essential for proper tissue formation and maintenance. We investigated autophagy, the process in which cells engulf cytoplasmic material in vesicles transported to lysosomes for degradation, in fibroblasts induced into a quiescent state through contact inhibition. Previous studies have shown increased autophagic vesicles in fibroblasts induced into quiescence by contact inhibition. However, our lab’s data showed that a key regulator of autophagy, TOR, is high in contact inhibited (CI) cells, making it unlikely these vesicles were generated by the canonical autophagy pathway. We hypothesized instead they may reflect a noncanonical autophagy pathway. To test this, we measured the extent of noncanonical autophagy in proliferating and quiescent fibroblasts using a pH dependent fluorescence from DQ-collagen that fluoresces when it encounters the lysosome. We found CI fibroblasts had higher rates of collagen digestion than proliferating fibroblasts. Further, using mouse embryonic fibroblasts with a mutation in the ATG16L1 protein, making them unable to perform noncanonical autophagy, we showed increased collagen digestion in CI fibroblasts requires the noncanonical autophagy pathway. This suggests CI fibroblasts rely on noncanonical autophagy to recycle the collagen receptor. The findings suggest noncanonical autophagy may be essential for wound healing. We next plan to investigate the importance of noncanonical autophagy in the context of fibrotic disease.

DOCK8 Deficiency is caused by large deletions in the DOCK8 gene, leading to recurrent infections on the skin and in the respiratory system. It is currently treated using allogeneic hematopoietic stem cell transplantation (HSCT) associated with life-threatening side effects. Lentiviral and adenoviral gene editing strategies are current techniques that are promising to restore DOCK8 function in a patient’s own stem cells. However, CRISPR/Cas9 site-specific editing techniques have not been well studied for large genes like DOCK8. Here I test adeno-associated virus serotype 6 (AAV6), Ad5.F35 adenovirus, and integrase deficient lentivirus (IDLV) in DOCK8 knockout (KO) cells to determine restoration of functional protein. Editing reagents were electroporated in KO Ramos B cells and evaluated for viability, fold expansion, genomic integration via ddPCR, and protein expression via flow cytometry. These reagents were well tolerated, inducing little to no toxicity on the cells. However, genomic integration of the cargo was low with no appreciable protein expression. Our preliminary results demonstrate that site-specific editing for large cargoes like DOCK8 is hindered by the size of donors. These initial tests of CRISPR/Cas9 editing in B cells allow for application of the same techniques to patient hematopoietic stem cells, the progenitor to all immune cells in which DOCK8 is expressed. Ultimately, this approach presents a key alternative to allogeneic HSCT as a much safer cure, significantly increasing patient life expectancy.

Mucus cells are a conserved cell type that secrete mucus onto epithelial surfaces, lubricating tissues and providing protection against pathogens. Mucus cells originate from stem-like basal cells in the epithelium. In larval zebrafish, basal cells divide, migrate through the intraepithelial space, and later insert themselves into the periderm layer where they stop moving and start secreting mucus. Determining their method of migration is an important next step in characterizing their development. Cell migration is classified by mesenchymal or amoeboid migration, both of which rely on actin filament mechanics to propel the cell forward. Mesenchymal migration is distinguished by elongated cell shape and slow migration, while amoeboid migration is marked by rounder cell shape and fast migration. Mesenchymal migratory cells often have lamellipodia, leading-edge protrusions that require branched actin. Arp2/3, a protein complex involved in actin filament branching, is required for lamellipodia formation, and can be inhibited by treatment with the drug CK-666. In repeated experiments, mucus cell precursors continued to migrate when treated with CK-666. These results suggest that they do not require lamellipodia, and may be utilizing an as-yet uncharacterized mode of migration. Next steps will use alternative pharmacological agents to identify molecular mechanisms driving cell motility. These studies will help better understand how mucus cell migration may play a role in development, immune defense, and various disease states.

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, fibrosis, synovial inflammation, and chronic pain. Current treatments primarily address symptoms and do not restore damaged joint tissue. Perivascular mesenchymal stem cell (MSC)-like populations, including pericytes and adventicytes, are of particular interest in OA because they may contribute to fibrosis, neo-angiogenesis, immune modulation, and mesenchymal differentiation within the diseased joint environment. This project aims to characterize these perivascular progenitor populations in human osteoarthritic knee tissue. Using immunohistochemistry and fluorescent microscopy, OA tissue sections are analyzed for vascular and perivascular marker expression to evaluate cell localization and distribution across tissue compartments. In parallel, fresh OA tissue is mechanically and enzymatically dissociated into single-cell suspensions for fluorescence-activated cell sorting (FACS) to isolate CD34-/CD146+ pericytes and CD34+/CD146- adventicytes while excluding non-target cell populations. Sorted cells are then seeded in culture and monitored for adherence, morphology, growth, and differentiation potential. Together, these approaches will help define the spatial distribution, phenotype, and functional properties of perivascular MSC-like cells in OA. This work may improve understanding of OA pathophysiology and identify progenitor populations with relevance for future regenerative therapies.

This abstract has been withheld from publication.