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Background 3D cell culture models have recently garnered increasing attention for replicating organ microarchitecture and eliciting in vivo-like responses, holding significant promise across various biological disciplines. Broadly, 3D cell culture encompasses organoids as well as single- and multicellular spheroids. While the latter have found successful applications in tumor research, there is a notable scarcity of standardized intestinal models for infection biology that mimic the microarchitecture of the intestine. Hence, this study aimed to develop structured multicellular intestinal spheroids (SMIS) specifically tailored for studying molecular basis of infection by intestinal pathogens. Results We have successfully engineered human SMIS comprising four relevant cell types, featuring a fibroblast core enveloped by an outer monolayer of enterocytes and goblet cells along with monocytic cells. These SMIS effectively emulate the in vivo architecture of the intestinal mucosal surface and manifest differentiated morphological characteristics, including the presence of microvilli, within a mere two days of culture. Through analysis of various differentiation factors, we have illustrated that these spheroids attain heightened levels of differentiation compared to 2D monolayers. Moreover, SMIS serve as an optimized intestinal infection model, surpassing the capabilities of traditional 2D cultures, and exhibit a regulatory pattern of immunological markers similar to in vivo infections after Campylobacter jejuni infection. Notably, our protocol extends beyond human spheroids, demonstrating adaptability to other species such as mice and pigs. Conclusion Based on the rapid attainment of enhanced differentiation states, coupled with the emergence of functional brush border features, increased cellular complexity, and replication of the intestinal mucosal microarchitecture, which allows for exposure studies via the medium, we are confident that our innovative SMIS model surpasses conventional cell culture methods as a superior model. Moreover, it offers advantages over stem cell-derived organoids due to scalability and standardization capabilities of the protocol. By showcasing differentiated morphological attributes, our model provides an optimal platform for diverse applications. Furthermore, the investigated differences of several immunological factors compared to monotypic monolayers after Campylobacter jejuni infection underline the refinement of our spheroid model, which closely mimics important features of in vivo infections. Keywords: Multilayered spheroid, 3D cell culture, Intestinal mucosa, Infection model, Author(s): Angelina Kraski[sup.1], PaweÅ MigdaÅ[sup.2], Robert Klopfleisch[sup.3], Clara Räckel[sup.1], Jutta Sharbati[sup.4], Markus M. Heimesaat[sup.5], Thomas Alter[sup.6], Carlos Hanisch[sup.7], Greta Gölz[sup.6], Ralf Einspanier[sup.1] and Soroush Sharbati[sup.1] Background For many years, cell [...]

Epithelial barriers are programmed for defense and repair but are also the site of long-term structural remodeling and disease. In general, this paradigm features epithelial stem cells (ESCs) that are called on to regenerate damaged tissues but can also be reprogrammed for detrimental remodeling. Here we identified a Wfdc21-dependent monocyte-derived dendritic cell (moDC) population that functioned as an early sentinel niche for basal ESC reprogramming in mouse models of epithelial injury after respiratory viral infection. Niche function depended on moDC delivery of ligand GPNMB to the basal ESC receptor CD44 so that properly timed antibody blockade of ligand or receptor provided long-lasting correction of reprogramming and broad disease phenotypes. These same control points worked directly in mouse and human basal ESC organoids. Together, the findings identify a mechanism to explain and modify what is otherwise a stereotyped but sometimes detrimental response to epithelial injury., Introduction Epithelial barriers maintain a fundamental responsibility to respond to injuries from the full range of environmental toxins and infectious agents. This role depends critically on carefully controlled growth and [...]

Introduction Skeletal stem cells (SSCs), as established by Paolo Bianco and colleagues, have the ability to generate colony-forming units and to differentiate into bone, cartilage, adipocyte, s and stromal cells [...], The periosteum contains skeletal stem/progenitor cells that contribute to bone fracture healing. However, the in vivo identity of periosteal skeletal stem cells (P-SSCs) remains unclear, and membrane protein markers of P-SSCs that facilitate tissue engineering are needed. Here, we identified integral membrane protein 2A (Itm2a) enriched in SSCs using single-cell transcriptomics. [Itm2a.sup.+] P-SSCs displayed clonal multipotency and self-renewal and sat at the apex of their differentiation hierarchy. Lineage-tracing experiments showed that Itm2a selectively labeled the periosteum and that [Itm2a.sup.+] cells were preferentially located in the outer fibrous layer of the periosteum. The [Itm2a.sup.+] cells rarely expressed CD34 or Osx, but expressed periosteal markers such as Ctsk, CD51, PDGFRA, Seal, and Gli1. [Itm2a.sup.+] P-SSCs contributed to osteoblasts, chondrocytes, and marrow stromal cells upon injury. Genetic lineage tracing using dual recombinases showed that Itm2a and Prrx1 lineage cells generated spatially separated subsets of chondrocytes and osteoblasts during fracture healing. Bone morphogenetic protein 2 (Bmp2) deficiency or ablation of [Itm2a.sup.+] P-SSCs resulted in defects in fracture healing. [ITM2A.sup.+] P-SSCs were also present in the human periosteum. Thus, our study identified a membrane protein marker that labels P-SSCs, providing an attractive target for drug and cellular therapy for skeletal disorders.

Introduction Metastases account for the vast majority of cancer-related deaths (1), however, why some cancers metastasize while others do not is poorly understood. Specific genetic alterations do not define metastatic [...], Metastasis is the leading cause of cancer-related deaths. It is unclear how intratumor heterogeneity (ITH) contributes to metastasis and how metastatic cells adapt to distant tissue environments. The study of these adaptations is challenged by the limited access to patient material and a lack of experimental models that appropriately recapitulate ITH. To investigate metastatic cell adaptations and the contribution of ITH to metastasis, we analyzed single-cell transcriptomes of matched primary tumors and metastases from patient-derived xenograft models of breast cancer. We found profound transcriptional differences between the primary tumor and metastatic cells. Primary tumors upregulated several metabolic genes, whereas motility pathway genes were upregulated in micrometastases, and stress response signaling was upregulated during progression. Additionally, we identified primary tumor gene signatures that were associated with increased metastatic potential and correlated with patient outcomes. Immune-regulatory control pathways were enriched in poorly metastatic primary tumors, whereas genes involved in epithelial-mesenchymal transition were upregulated in highly metastatic tumors. We found that ITH was dominated by epithelial-mesenchymal plasticity (EMP), which presented as a dynamic continuum with intermediate EMP cell states characterized by specific genes such as CRYAB and S100A2. Elevated expression of an intermediate EMP signature correlated with worse patient outcomes. Our findings identified inhibition of the intermediate EMP cell state as a potential therapeutic target to block metastasis.

Craniofacial anomalies, especially midline facial defects, are among the most common birth defects in patients and are associated with increased mortality or require lifelong treatment. During mammalian embryogenesis, specific instructions arising at genetic, signaling, and metabolic levels are important for stem cell behaviors and fate determination, but how these functionally relevant mechanisms are coordinated to regulate craniofacial morphogenesis remain unknown. Here, we report that bone morphogenetic protein (BMP) signaling in cranial neural crest cells (CNCCs) is critical for glycolytic lactate production and subsequent epigenetic histone lactylation, thereby dictating craniofacial morphogenesis. Elevated BMP signaling in CNCCs through constitutively activated ACVR1 (ca-ACVR1) suppressed glycolytic activity and blocked lactate production via a p53- dependent process that resulted in severe midline facial defects. By modulating epigenetic remodeling, BMP signaling- dependent lactate generation drove histone lactylation levels to alter essential genes of Pdgfra, thus regulating CNCC behavior in vitro as well as in vivo. These findings define an axis wherein BMP signaling controls a metabolic/epigenetic cascade to direct craniofacial morphogenesis, thus providing a conceptual framework for understanding the interaction between genetic and metabolic cues operative during embryonic development. These findings indicate potential preventive strategies of congenital craniofacial birth defects via modulating metabolic-driven histone lactylation., Introduction Craniofacial anomalies, especially midline facial defects, are among the most common birth defects found in patient populations whose appearance are either associated with increased mortality or require lifelong treatment [...]