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1. Engineered epithelial curvature controls Paneth cell localization in intestinal organoids

   Yavitt, FM; Khang, A; Bera, K; McNally, D L; Blatchley, MR; Gallagher, AP; Klein, OD; Castillo-Azofeifa, D; Dempsey, PJ; Anseth, KS (April 2025, Cell Biomaterials)

   The cellular organization within organoid models is important to regulate tissue-specific function, yet few engineering approaches can control or direct cellular organization. Here, a photodegradable hydrogel is used to create softened regions that direct crypt formation within intestinal organoids, where the dimensions of the photosoftened regions generate predictable and defined crypt architectures. Guided by in vivo metrics of crypt morphology, this photopatterning method is used to control the width and length of in vitro organoid crypts, which ultimately defines the curvature of the epithelium. By tracking expression of differentiated Paneth-cell markers in real time, we show that epithelial curvature directs the localization of Paneth cells within engineered crypts, providing user-directed control over organoid functionality. We anticipate that our improved control over organoid architecture and thus Paneth-cell localization will lead to more consistent in vitro organoid models for both mechanistic studies and translational applications. 

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2. Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine

   https://doi.org/10.1016/j.immuni.2020.07.010  

   Yu, Shiyan; Balasubramanian, Iyshwarya; Laubitz, Daniel; Tong, Kevin; Bandyopadhyay, Sheila; Lin, Xiang; Flores, Juan; Singh, Rajbir; Liu, Yue; Macazana, Carlos; et al (August 2020, Immunity)

   null (Ed.)

   Paneth cells are the primary source of C-type lysozyme, a b-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn’s disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1
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   hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD. 

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3. Intracellular pH dynamics regulates intestinal stem cell lineage specification

   https://doi.org/10.1038/s41467-023-39312-9  

   Liu, Yi; Reyes, Efren; Castillo-Azofeifa, David; Klein, Ophir D.; Nystul, Todd; Barber, Diane L. (June 2023, Nature Communications)

   Abstract Intracellular pH dynamics is increasingly recognized to regulate myriad cell behaviors. We report a finding that intracellular pH dynamics also regulates adult stem cell lineage specification. We identify an intracellular pH gradient in mouse small intestinal crypts, lowest in crypt stem cells and increasing along the crypt column. Disrupting this gradient by inhibiting H⁺efflux by Na⁺/H⁺exchanger 1 abolishes crypt budding and blocks differentiation of Paneth cells, which are rescued with exogenous WNT. Using single-cell RNA sequencing and lineage tracing we demonstrate that intracellular pH dynamics acts downstream of ATOH1, with increased pH promoting differentiation toward the secretory lineage. Our findings indicate that an increase in pH is required for the lineage specification that contributes to crypt maintenance, establishing a role for intracellular pH dynamics in cell fate decisions within an adult stem cell lineage. 

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4. Brain Tissue-Derived Extracellular Vesicle Mediated Therapy in the Neonatal Ischemic Brain

   https://doi.org/10.3390/ijms23020620  

   Nguyen, Nam Phuong; Helmbrecht, Hawley; Ye, Ziming; Adebayo, Tolulope; Hashi, Najma; Doan, My-Anh; Nance, Elizabeth (January 2022, International Journal of Molecular Sciences)

   Hypoxic-Ischemic Encephalopathy (HIE) in the brain is the leading cause of morbidity and mortality in neonates and can lead to irreparable tissue damage and cognition. Thus, investigating key mediators of the HI response to identify points of therapeutic intervention has significant clinical potential. Brain repair after HI requires highly coordinated injury responses mediated by cell-derived extracellular vesicles (EVs). Studies show that stem cell-derived EVs attenuate the injury response in ischemic models by releasing neuroprotective, neurogenic, and anti-inflammatory factors. In contrast to 2D cell cultures, we successfully isolated and characterized EVs from whole brain rat tissue (BEV) to study the therapeutic potential of endogenous EVs. We showed that BEVs decrease cytotoxicity in an ex vivo oxygen glucose deprivation (OGD) brain slice model of HI in a dose- and time-dependent manner. The minimum therapeutic dosage was determined to be 25 μg BEVs with a therapeutic application time window of 4–24 h post-injury. At this therapeutic dosage, BEV treatment increased anti-inflammatory cytokine expression. The morphology of microglia was also observed to shift from an amoeboid, inflammatory phenotype to a restorative, anti-inflammatory phenotype between 24–48 h of BEV exposure after OGD injury, indicating a shift in phenotype following BEV treatment. These results demonstrate the use of OWH brain slices to facilitate understanding of BEV activity and therapeutic potential in complex brain pathologies for treating neurological injury in neonates. 

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5. Trellis tree-based analysis reveals stromal regulation of patient-derived organoid drug responses

   https://doi.org/10.1016/j.cell.2023.11.005  

   Ramos_Zapatero, Maria; Tong, Alexander; Opzoomer, James W; O’Sullivan, Rhianna; Cardoso_Rodriguez, Ferran; Sufi, Jahangir; Vlckova, Petra; Nattress, Callum; Qin, Xiao; Claus, Jeroen; et al (December 2023, Cell)

   Patient-derived organoids (PDOs) can model personalized therapy responses; however, current screening technologies cannot reveal drug response mechanisms or how tumor microenvironment cells alter therapeutic performance. To address this, we developed a highly multiplexed mass cytometry platform to measure post- translational modification (PTM) signaling, DNA damage, cell-cycle activity, and apoptosis in >2,500 colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies at single-cell resolution. To compare patient- and microenvironment-specific drug responses in thousands of single-cell da- tasets, we developed ‘‘Trellis’’—a highly scalable, tree-based treatment effect analysis method. Trellis single- cell screening revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is rarer, patient-specific, and aligns with cancer cell PTM signaling. We find that CAFs can regulate PDO plasticity—shifting proliferative colonic stem cells (proCSCs) to slow-cycling revival colonic stem cells (revCSCs) to protect cancer cells from chemotherapy. 

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