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Myocilin is a secreted glycoprotein that is expressed in ocular and non-ocular tissues. Mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. Here we report that myocilin is expressed in bone marrow-derived mesenchymal stem cells (MSCs) and plays a role in their differentiation into osteoblasts in vitro and in osteogenesis in vivo. Expression of myocilin was detected in MSCs derived from mouse,rat,and human bone marrow,with human MSCs exhibiting the highest level of myocilin expression. Expression of myocilin rose during the course of human MSC differentiation into osteoblasts but not into adipocytes,and treatment with exogenous myocilin further enhanced osteogenesis. MSCs derived from Myoc-null mice had a reduced ability to differentiate into the osteoblastic lineage,which was partially rescued by exogenous extracellular myocilin treatment. Myocilin also stimulated osteogenic differentiation of wild-type MSCs,which was associated with activation of the p38,Erk1/2,and JNK MAP kinase signaling pathways as well as up-regulated expression of the osteogenic transcription factors Runx2 and Dlx5. Finally,cortical bone thickness and trabecular volume,as well as the expression level of osteopontin,a known factor of bone remodeling and osteoblast differentiation,were reduced dramatically in the femurs of Myoc-null mice compared with wild-type mice. These data suggest that myocilin should be considered as a target for improving the bone regenerative potential of MSCs and may identify a new role for myocilin in bone formation and/or maintenance in vivo. View Publication

Neurogenin3+ (Ngn3+) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin,glucagon,somatostatin,pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach,where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors,but the lineage and regulators of pancreatic gastrin+ cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin+ cells in the developing pancreas co-express glucagon,ghrelin or pancreatic polypeptide,but many gastrin+ cells do not express any other islet hormone. Pancreatic gastrin+ cells express the transcription factors Nkx6.1,Nkx2.2 and low levels of Pdx1,and derive from Ngn3+ endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3,Nkx2.2,NeuroD1 and Arx,but not Pax4 or Pax6. Finally,gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus,gastrin+ cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells. View Publication

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity,immunodeficiencies,and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies,which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-$\gamma$ (IFN-$\gamma$) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization,revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits,revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions,which could inform the design of immunotherapies. View Publication

Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However,generation of human AID cell lines is challenging,especially in human embryonic stem cells (hESCs). Here,we develop HiHo-AID2,a streamlined procedure for rapid,one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines,including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03187-w. View Publication

SummaryFocusing on the early stages of Alzheimer’s disease (AD) holds great promise. However,the specific events in neural cells preceding AD onset remain elusive. To address this,we utilized human-induced pluripotent stem cells carrying APPswe mutation to explore the initial changes associated with AD progression. We observed enhanced neural activity and early neuronal differentiation in APPswe cerebral organoids cultured for one month. This phenomenon was also evident when neural progenitor cells (NPCs) were differentiated into neurons. Furthermore,transcriptomic analyses of NPCs and neurons confirmed altered expression of neurogenesis-related genes in APPswe NPCs. We also found that the upregulation of reactive oxygen species (ROS) is crucial for early neuronal differentiation in these cells. In addition,APPswe neurons remained immature after initial differentiation with increased susceptibility to toxicity,providing valuable insights into the premature exit from the neural progenitor state and the increased vulnerability of neural cells in AD. Graphical abstract Highlights•APPswe organoids show increased neural activity and early differentiation•Enhanced ROS levels are necessary but insufficient to accelerate differentiation•Transcriptome analysis of APPswe NPCs shows gene expression shift to differentiation•Premature neural cells with APPswe exhibit increased vulnerability to toxicity Molecular biology; Neuroscience; Cell biology View Publication

The role of the bone marrow (BM) microenvironment in regulating the antitumor immune response in Waldenstrom macroglobulinemia (WM) remains poorly understood. Here we transcriptionally and phenotypically profiled non-malignant (CD19- CD138-) BM cells from WM patients with a focus on myeloid derived suppressive cells (MDSCs) to provide a deeper understanding of their role in WM. We found that HLA-DRlowCD11b+CD33+ MDSCs were significantly increased in WM patients as compared to normal controls,with an expansion of predominantly polymorphonuclear (PMN)-MDSCs. Single-cell immunogenomic profiling of WM MDSCs identified an immune-suppressive gene signature with upregulated inflammatory pathways associated with interferon and tumor necrosis factor (TNF) signaling. Gene signatures associated with an inflammatory and immune suppressive environment were predominately expressed in PMN-MDSCs. In vitro,WM PMN-MDSCs demonstrated robust T-cell suppression and their viability and expansion was notably enhanced by granulocyte colony stimulating factor (G-CSF) and TNFα. Furthermore,BM malignant B-cells attracted PMN-MDSCs to a greater degree than monocytic MDSCs. Collectively,these data suggest that malignant WM B cells actively recruit PMN-MDSCs which promote an immunosuppressive BM microenvironment through a direct T cell inhibition,while release of G-CSF/TNFα in the microenvironment further promotes PMN-MDSC expansion and in turn immune suppression. Targeting PMN-MDSCs may therefore represent a potential therapeutic strategy in patients with WM. View Publication

Aim of work was to locate a simple,reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited,namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size,pore orientation,porosity,and pore distribution were produced using two different techniques,such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity,seeding efficiency,and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method,as these conditions successfully improved the cellularization of polymeric patches. Furthermore,the investigation provided interesting information on patches' stability in physiological simulating experimental conditions. Considering the in vitro results,it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover,the protocol turned out to be simple,repeatable,and reproducible. View Publication

Cancer stem cells (CSC) are purported to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of CSCs; however,the molecular determinants of this process remain poorly understood. Here we show that the transcriptional coactivator YAP1 is a major determinant of CSC properties in nontransformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSC properties. Conversely,shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenicity in vivo. The small-molecule inhibitor of YAP1,verteporfin,significantly blocks CSC properties in cells with high YAP1 and a high proportion of ALDH1(+). Our findings identify YAP1-driven SOX9 expression as a critical event in the acquisition of CSC properties,suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population. View Publication

Detection and characterization of rare circulating tumor cells (CTCs) in patients' blood is important for the diagnosis and monitoring of cancer. The traditional way of counting CTCs via fluorescent images requires a series of tedious experimental procedures and often impacts the viability of cells. Here we present a method for label-free detection of CTCs from patient blood samples,by taking advantage of data analysis of bright field microscopy images. The approach uses the convolutional neural network,a powerful image classification and machine learning algorithm to perform label-free classification of cells detected in microscopic images of patient blood samples containing white blood cells and CTCs. It requires minimal data pre-processing and has an easy experimental setup. Through our experiments,we show that our method can achieve high accuracy on the identification of rare CTCs without the need for advanced devices or expert users,thus providing a faster and simpler way for counting and identifying CTCs. With more data becoming available in the future,the machine learning model can be further improved and can serve as an accurate and easy-to-use tool for CTC analysis. View Publication

SF3B1,a component of the U2 snRNP pre-mRNA splicing factor,plays a critical role in splicing and is frequently mutated in cancer,particularly hematologic malignancies. We investigated the effects of the most common SF3B1 mutation,heterozygous substitution of Lysine 700 to Glutamate (K700E),in human embryonic stem cells (hESC),using CRISPR-Cas9 to generate heterozygous SF3B1K700E clones. Interestingly,we observed the upregulation of several key transcription regulators associated with hematopoiesis and a broad range of immune genes in SF3B1K700E hESCs. Despite differences in the transcriptional and splicing profiles between hESC and myelodysplastic syndrome (MDS) cells harboring the SF3B1K700E mutation,several common immune gene programs were identified in both cell types. To elucidate the molecular mechanisms underlying dysregulated gene expression in SF3B1K700E hESCs,we mapped actively engaged RNA polymerase II (RNA Pol II) using Precision Run-On sequencing (PRO-seq). These analyses revealed that the SF3B1K700E mutation alters RNA Pol II elongation properties. Specifically,we observed a general increase in pause release in SF3B1K700E hESCs,consistent with recent work in leukemia cells suggesting that the SF3B1K700E mutation affects early transcription elongation. Taken together,our study identifies several candidate genes that could contribute to the SF3B1 mutated phenotype and clarifies the role for the U2 snRNP and pre-spliceosome assembly on transcription by RNA Pol II. Further,our data suggest that mutations of SF3B1 impact immune gene expression independent of cell type,providing new insights into the role of SF3B1K700E in hematologic malignancies. View Publication

Antibiotic-resistant infections remain a major challenge in cystic fibrosis (CF),where chronic Pseudomonas aeruginosa colonization drives lung infection. The overexpression of adhesion-related proteins and extracellular matrix components,including fibronectin (Fn),facilitates bacterial colonization. Recent evidence identifies the RNA-binding protein Human Antigen R (HuR) as a key regulator of this process,as it stabilizes Vav3 mRNA,promoting Fn deposition and the formation of bacterial docking platforms. Here,we report the synthesis,optimization,and functional evaluation of the HuR-targeted small-molecule (2S,3S)-BOPC1. Functional assays in CF human airway epithelial cells demonstrated that (2S,3S)-BOPC1 significantly reduced P. aeruginosa adhesion in a dose-dependent manner without detectable cytotoxic effects. These findings provide the first evidence that targeting HuR can disrupt the HuR–Vav3–Fn axis,reducing bacterial attachment. This host-directed approach represents a promising strategy to prevent chronic infections in CF without promoting antibiotic resistance. View Publication