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Mantle cell lymphoma (MCL) is a clinically aggressive and incurable form of non-Hodgkin lymphoma with very heterogeneous clinical and biological behaviors. Dysregulation of RNA splicing machinery is common in various types of cancer,including hematologic malignancies,and is associated with cancer progression. However,whether and how splicing factors,the spliceosome components responsible for pre-mRNA splicing,regulate MCL aggressive behaviors remain largely unknown. Bioinformatics analyses were employed to profile the mRNA expression of two key families of splicing factors,that are serine/arginine-rich (SR) and heterogenous nuclear ribonucleoprotein (hnRNP) families,in clinical specimens of MCL patients in comparison to normal B cells. Functional roles of splicing factors in MCL aggressive phenotypes defined as hallmarks of cancer were investigated in MCL cell lines. Kaplan–Meier survival analyses were performed to determine the clinical significance of splicing factors according to their gene expression level. Depletion of SRSF1,hnRNP F,and PTBP1,which are highly expressed in MCL clinical specimens,by CRISPR/Cas9 system significantly inhibit cell growth and proliferation,motility,and angiogenesis of MCL cells. SRSF1,hnRNP F (for Z-138 cells),and PTBP1 were found to mediate BTZ sensitivity in MCL cells,in agreement with an increase in caspase-3 activation and the occurrence of splicing events that favor the expression of pro-apoptotic isoforms of apoptosis regulatory genes. We also found that depletion of SRSF1,hnRNP F,and PTBP1 induces the expression of autophagy-related genes and the accumulation of autophagic vacuoles,which may act as one of the tumor-suppressive mechanisms. Survival analyses revealed that co-high expression of SRSF1,HNRNPF,or PTBP1 and oncogenic MYC predicts poor clinical outcomes in MCL patients. Our data describe the clinical significance of aberrant SRSF1,hnRNP F,and PTBP1 in MCL and their tumor-promoting roles via the regulation of cancer hallmarks,which could be important in understanding MCL pathogenesis and therapeutic development. View Publication

Over the past decade,significant advancements have been made in understanding the developmental mechanisms involved in human gastrointestinal formation,with organoids emerging as key experimental models. These three‐dimensional in vitro cellular structures mimic the organization and functions of various gut regions,providing a powerful tool for research. By replicating critical stages of gut development,we can now direct the differentiation of cells into specific gastrointestinal tissues. In this protocol,we outline how to generate two types of organoids derived from human pluripotent stem cells (hPSCs): human intestinal organoids (HIOs) and human colonic organoids (HCOs). First,we induce definitive endoderm formation to produce these organoids and specify midgut/hindgut tissues. Three‐dimensional spheroids form spontaneously,can be collected,embedded in an extracellular matrix,and cultured over time. During this phase,the organoid epithelium develops,supported by a mesenchymal layer that promotes maturation and differentiation. After a month of culture,HIOs and HCOs reach a developmental and maturation stage comparable to that of the human fetal intestine. These organoids can be used to study human gastrointestinal development,model diseases,and test therapeutic agents. Human pluripotent stem cells can be guided through a stepwise differentiation process to produce self‐organizing intestinal and colonic organoids. The resulting organoids recapitulate fetal‐stage epithelial and mesenchymal organization,offering a powerful model to explore human gastrointestinal development and its disorders. View Publication

The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However,such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here,we present SNIPE,a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells,which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations. Genome editing often requires marker genes for selection of edited cells. Here,the authors present SNIPE,a marker-free method that selects cells based on DNA sequence,enabling precise enrichment of edited cells and applications from evolutionary research to the elimination of cancer cells. View Publication

Older adults have decreased vaccine efficacy,but the adjuvanted recombinant VZV-gE zoster vaccine (RZV) is highly efficacious. We investigated memory-like innate immune responses after RZV and after the zoster vaccine live (ZVL),which is much less efficacious. RZV increased NK,monocyte,and DC activation in response to in vitro VZV-gE stimulation for up to 5 years post-vaccination,while ZVL increased only DC responses to VZV for up to 90 days. In purified monocyte and NK cell cocultures,RZV recipients showed increased responses to VZV-gE,HCMV and HSV antigenic stimulation post-vaccination. ATAC-seq analysis of purified monocytes revealed decreased accessibility in areas of the TGFβ1 gene. scRNA-seq and immunoproteomics confirmed decreased TGFβ1 transcription and translation,respectively. Exogenous supplementation and inhibition of TGFβ1 modulated in vitro monocyte responses to VZV-gE. In conclusion,RZV generated homologous (VZV-gE) and heterologous (HCMV,HSV) trained immunity in monocytes through genomic repression of the regulatory cytokine TGFβ-1. Cytokine modulation may represent a novel mechanism of generating trained immunity in myeloid cells. Author summaryOlder adults have decreased vaccine efficacy,but the adjuvanted recombinant varicella zoster virus (VZV)-gE zoster vaccine (RZV; Shingrix™) is highly efficacious. We investigated memory-like innate immune responses after RZV and after the zoster vaccine live (ZVL; Zostavax™),which is much less efficacious than RZV. We found that RZV increased the functionality of several innate immune cell subsets against VZV-gE other herpesviruses. The increase in functionality was associated with decreased production of the inhibitory cytokine TGFβ1,which may have resulted from decreased ability to use the TGFβ1 gene as a template for the synthesis of its product. We concluded that RZV generated homologous (VZV-gE) and heterologous (other herpesviruses) memory-like responses in innate immune cell subsets through genomic repression of the regulatory cytokine TGFβ-1. View Publication