SUMMARY microRNAs regulate developmental cell fate decisions, tissue homeostasis and oncogenesis in distinct ways relative to proteins. Here, we show that the tumor suppressor microRNA miR-34a is a cell fate determinant in early stage dividing colon cancer stem cells (CCSCs). In pair-cell assays, miR34a distributes at high levels in differentiating progeny, while low levels of miR34a demarcate self renewing CCSCs. Moreover, miR34a loss of function and gain of function alters the balance between self-renewal and differentiation both in vitro and in vivo. Mechanistically, miR34a sequesters Notch1 mRNA to generate a sharp threshold response where a bimodal Notch signal specifies the choice between self-renewal versus differentiation. In contrast, the canonical cell fate determinant Numb regulates Notch levels in a continuously graded manner. Taken together, our findings highlight a unique microRNA regulated mechanism that converts noisy input into a toggle switch for robust cell fate decisions in CCSCs.

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Lynch syndrome, also known as hereditary nonpolyposis colon cancer (HNPCC), is the most common known genetic syndrome for colorectal cancer (CRC). MLH1/MSH2 mutations underlie approximately 90% of Lynch syndrome families. A total of 24% of these mutations are missense. Interpreting missense variation is extremely challenging. We have therefore developed multivariate analysis of protein polymorphisms-mismatch repair (MAPP-MMR), a bioinformatic algorithm that effectively classifies MLH1/MSH2 deleterious and neutral missense variants. We compiled a large database (n>300) of MLH1/MSH2 missense variants with associated clinical and molecular characteristics. We divided this database into nonoverlapping training and validation sets and tested MAPP-MMR. MAPP-MMR significantly outperformed other missense variant classification algorithms (sensitivity, 94%; specificity, 96%; positive predictive value [PPV] 98%; negative predictive value [NPV], 89%), such as SIFT and PolyPhen. MAPP-MMR is an effective bioinformatic tool for missense variant interpretation that accurately distinguishes MLH1/MSH2 deleterious variants from neutral variants.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration 1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2) 3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens 4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting. Endothelial cells (ECs) in zonated capillaries sustain tissue-specific homeostasis and supply angiocrine factors to guide organ regeneration 1,2. By contrast, maladaptation of ECs contributes to fibrosis and tumour progression 6,7. The mechanism(s) by which ECs acquire adaptive tissue-specific heterogeneity or maladapt within the scarred tissues or tumour microenvironment are unknown. Identifying the molecular determinants of vascular heterogeneity requires the generation of malleable and perfusable vascular networks that are

E. coli AlkB has been intensively studied since 1983, but the in vivo roles of its mammalian homologue Alkbh1 are unknown. We, therefore, created null mice for Alkbh1. Alkbh1 mRNA is expressed at highest levels in the trophoblast lineages of the developing placenta.