Levi, Y. | bioRxiv Science

bioRxiv2024

Human adipose-derived mesenchymal stromal cells improved wound healing in enterocutaneous fistulizing disease mouse model

A significant complication of Crohns disease is the formation of perianal fistulas. While local application of human adipose tissue-derived mesenchymal stromal cells (AT-MSC INN Darvadstrocel, Takeda) is an approved therapy for this condition, challenges in studying human clinical tissue and the absence of a robust experimental model have hindered deeper understanding of the preclinical efficacy and mechanisms behind the therapy. We have developed a model that closely mimics the clinical scenario, using human gut tissue transplanted subcutaneously into SCID mice. In this model, enterocutaneous fistulas are reliably induced by combining imiquimod-induced psoriatic dermatitis above the transplant with systemic lipopolysaccharide (LPS), a major component of the bacterial cell wall. Similar to clinical observations, local application of AT-MSC significantly improved fistula wound healing and re-epithelialization. This model system enabled comprehensive tissue harvesting for histopathological analysis, spatial transcriptomics, and protein profiling. Our findings demonstrate that AT-MSC applied around the fistula wound survive for up to three weeks, migrating into the fistula tract and further into the inflamed human gut tissue. Within the fistula tract, AT-MSCs acquired immune-active properties, with increased expression of SOD2 and CCL2, and were associated with a substantial population of M2 macrophages. In contrast, AT-MSC located in healthy tissue near the fistula remained stationary, adopting a fibroblast-like phenotype within a collagen-rich extracellular matrix. These preclinical results support the safety and efficacy of AT-MSC for treating fistulizing gut disease. Furthermore, our data suggest that the inflamed fistula tract acts as a scaffold promoting AT-MSC activity and migration. Enhancing SOD2 and CCL2 expression through pre-activation or genetic modification may further improve the therapeutic potential of these cells.

pathology↗

bioRxiv2024

Genetically encoded biosensor for fluorescence lifetime imaging of PTEN dynamics in the intact brain

The phosphatase and tensin homolog (PTEN) is a vital signaling protein which maintains an inhibitory brake that is critical for cellular metabolism, proliferation, and growth. The importance of PTEN signaling is evident from the broad spectrum of human pathologies associated with its loss of function. Moreover, loss or gain of PTEN function in animal models leads to aberrant cellular morphology, function, and metabolic regulation. However, despite the important role of PTEN signaling, there is currently no method to dynamically monitor its activity with cellular specificity within intact biological systems. Here, we describe the development of a novel PTEN biosensor, optimized for two-photon fluorescence lifetime imaging microscopy (2pFLIM). This biosensor is designed to measure PTEN activity within intact cells, tissues, and organisms. Our approach is based on monitoring FRET-dependent changes in PTEN conformation, which serves as a proxy for the activity state in living cells. We identify a point mutation that allow us to express this biosensor with minimal interference to endogenous PTEN signaling and cellular function. We demonstrate the utility of imaging PTEN signaling in cell lines, developing C. elegans, and in the living mouse brain. To complement this approach, we developed a red-shifted PTEN sensor variant that permits simultaneous imaging with GFP-based sensors. Finally, we use in vivo PTEN imaging in the mouse brain to identify cell-type specific dynamics of PTEN activity in excitatory and inhibitory cortical cells. In summary, our approach enables dynamic imaging of PTEN activity in vivo with unprecedented spatial and temporal resolution.

neuroscience↗