Time-resolved fate mapping identifies the intestinal upper crypt zone as an origin of Lgr5+ crypt base columnar cells.

In the prevailing model, Lgr5+ cells are the only intestinal stem cells (ISCs) that sustain homeostatic epithelial regeneration by upward migration of progeny through elusive upper crypt transit-amplifying (TA) intermediates. Here, we identify a proliferative upper crypt population marked by Fgfbp1, in the location of putative TA cells, that is transcriptionally distinct from Lgr5+ cells. Using a kinetic reporter for time-resolved fate mapping and Fgfbp1-CreERT2 lineage tracing, we establish that Fgfbp1+ cells are multi-potent and give rise to Lgr5+ cells, consistent with their ISC function. Fgfbp1+ cells also sustain epithelial regeneration following Lgr5+ cell depletion. We demonstrate that FGFBP1, produced by the upper crypt cells, is an essential factor for crypt proliferation and epithelial homeostasis. Our findings support a model in which tissue regeneration originates from upper crypt Fgfbp1+ cells that generate progeny propagating bi-directionally along the crypt-villus axis and serve as a source of Lgr5+ cells in the crypt base.

Single-Cell Transcriptional Profiling of the Intestinal Epithelium.

Emerging single-cell technologies, like single-cell RNA sequencing (scRNA-seq), enable the study of heterogeneous biological systems at cellular resolution. By profiling the set of expressed transcripts in each cell, single-cell transcriptomics has allowed for the cataloging of the cellular constituents of multiple organs and tissues, both in health and disease. In addition, these technologies have provided mechanistic insights into cellular function, cell state transitions, developmental trajectories and lineage relationships, as well as helped to dissect complex, population-level responses to environmental perturbations. scRNA-seq is particularly useful for characterizing the intestinal epithelium because it is a dynamic, rapidly self-renewing tissue comprised of more than a dozen specialized cell types. Here we discuss the fundamentals of single-cell transcriptomics of the murine small intestinal epithelium. We review the principles of proper experimental design and provide methods for the dissociation of the small intestinal epithelium into single cells followed by fluorescence-activated cell sorting (FACS) and for scRNA-seq using the 10× Genomics Chromium platform.

Single-Cell Studies of Intestinal Stem Cell Heterogeneity During Homeostasis and Regeneration.

Single-cell RNA-sequencing (scRNA-seq) provides a unique opportunity to study heterogeneous cell populations within tissues, including the intestinal epithelium, to gain detailed molecular insights into their biology. Many new putative markers of intestinal stem cells and their progeny have been described using single-cell transcriptomics, which has contributed to the identification of novel subpopulations of mature cell types and insight into their developmental trajectories. This approach has revealed tremendous cellular heterogeneity within the intestinal epithelium that is concordant with its diverse and multifaceted functions. We discuss the function of these subpopulations during tissue homeostasis, as well as putative subpopulations with inducible regenerative potential following tissue injury.

Induction of apoptosis via proteasome inhibition in leukemia/lymphoma cells by two potent piperidones.

PURPOSE: Previously, compounds containing a piperidone structure have been shown to be highly cytotoxic to cancer cells. Recently, we found that the piperidone compound P2 exhibits a potent anti-neoplastic activity against human breast cancer-derived cells. Here, we aimed to evaluate two piperidone compounds, P1 and P2, for their potential anti-neoplastic activity against human leukemia/lymphoma-derived cells. METHODS: Cytotoxicity and apoptosis induction were evaluated using MTS, annexin V-FITC/PI and mitochondrial membrane potential polychromatic assays to confirm the mode of action of the piperidone compounds. The effects of compound P1 and P2 treatment on gene expression were assessed using AmpliSeq analysis and, subsequently, confirmed by RT-qPCR and Western blotting. RESULTS: We found that the two related piperidone compounds P1 and P2 selectively killed the leukemia/lymphoma cells tested at nanomolar concentrations through induction of the intrinsic apoptotic pathway, as demonstrated by mitochondrial depolarization and caspase-3 activation. AmpliSeq-based transcriptome analyses of the effects of compounds P1 and P2 on HL-60 acute leukemia cells revealed a differential expression of hundreds of genes, 358 of which were found to be affected by both. Additional pathway analyses revealed that a significant number of the common genes were related to the unfolded protein response, implying a possible role of the two compounds in the induction of proteotoxic stress. Subsequent analyses of the transcriptome data revealed that P1 and P2 induced similar gene expression alterations as other well-known proteasome inhibitors. Finally, we found that Noxa, an important mediator of the activity of proteasome inhibitors, was significantly upregulated at both the mRNA and protein levels, indicating a possible role in the cytotoxic mechanism induced by P1 and P2. CONCLUSIONS: Our data indicate that the cytotoxic activity of P1 and P2 on leukemia/lymphoma cells is mediated by proteasome inhibition, leading to activation of pro-apoptotic pathways.

Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity.

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewing's family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression.

PURPOSE: To determine the pharmacokinetics and the antitumor activity in pediatric cancer models of MM-398, a nanoliposomal irinotecan (nal-IRI). EXPERIMENTAL DESIGN: Mouse plasma and tissue pharmacokinetics of nal-IRI and the current clinical formulation of irinotecan were characterized. In vivo activity of irinotecan and nal-IRI was compared in xenograft models (3 each in nu/nu mice) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 expression was assessed by Affymetrix HuEx arrays, Taqman RT-PCR, and immunoblotting. RESULTS: Plasma and tumor concentrations of irinotecan and SN-38 (active metabolite) were approximately 10-fold higher for nal-IRI than for irinotecan. Two doses of NAL-IRI (10 mg/kg/dose) achieved complete responses maintained for >100 days in 24 of 27 EFT-xenografted mice. Event-free survival for mice with RMS and NB was significantly shorter than for EFT. High SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents; median SLFN11 mRNA expression was >100-fold greater in both EFT cell lines and primary tumors compared with NB or RMS cell lines or primary tumors. Cytotoxicity of SN-38 inversely correlated with SLFN11 mRNA expression in 20 EFT cell lines. CONCLUSIONS: In pediatric solid tumor xenografts, nal-IRI demonstrated higher systemic and tumor exposures to SN-38 and improved antitumor activity compared with the current clinical formulation of irinotecan. Clinical studies of nal-IRI in pediatric solid tumors (especially EFT) and correlative studies to determine if SLFN11 expression can serve as a biomarker to predict nal-IRI clinical activity are warranted.