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Mammary and extramammary Paget's Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways - including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.

A certain number of epithelial cells in intestinal crypts are DNA damage resistant and contribute to regeneration. However, the cellular mechanism underlying intestinal regeneration remains unclear. Using lineage tracing, we show that cells marked by an Msi1 reporter (Msi1+) are right above Lgr5high cells in intestinal crypts and exhibit DNA damage resistance. Single-cell RNA sequencing reveals that the Msi1+ cells are heterogeneous with the majority being intestinal stem cells (ISCs). The DNA damage-resistant subpopulation of Msi1+ cells is characterized by low-to-negative Lgr5 expression and is more rapidly cycling than Lgr5high radiosensitive crypt base columnar stem cells (CBCs). This enables an efficient repopulation of the intestinal epithelium at early stage when Lgr5high cells are not emerging. Furthermore, relative to CBCs, Msi1+ cells preferentially produce Paneth cells during homeostasis and upon radiation repair. Together, we demonstrate that the DNA damage-resistant Msi1+ cells are cycling ISCs that maintain and regenerate the intestinal epithelium.

Cerebral cavernous malformation (CCM) is a genetic, cerebrovascular disease. Familial CCM is caused by genetic mutations in KRIT1, CCM2, or PDCD10 Disease onset is earlier and more severe in individuals with PDCD10 mutations. Recent studies have shown that lesions arise from excess mitogen-activated protein kinase kinase kinase 3 (MEKK3) signaling downstream of Toll-like receptor 4 (TLR4) stimulation by lipopolysaccharide derived from the gut microbiome. These findings suggest a gut-brain CCM disease axis but fail to define it or explain the poor prognosis of patients with PDCD10 mutations. Here, we demonstrate that the gut barrier is a primary determinant of CCM disease course, independent of microbiome configuration, that explains the increased severity of CCM disease associated with PDCD10 deficiency. Chemical disruption of the gut barrier with dextran sulfate sodium augments CCM formation in a mouse model, as does genetic loss of Pdcd10, but not Krit1, in gut epithelial cells. Loss of gut epithelial Pdcd10 results in disruption of the colonic mucosal barrier. Accordingly, loss of Mucin-2 or exposure to dietary emulsifiers that reduce the mucus barrier increases CCM burden analogous to loss of Pdcd10 in the gut epithelium. Last, we show that treatment with dexamethasone potently inhibits CCM formation in mice because of the combined effect of action at both brain endothelial cells and gut epithelial cells. These studies define a gut-brain disease axis in an experimental model of CCM in which a single gene is required for two critical components: gut epithelial function and brain endothelial signaling.

Alternative splicing (AS) plays a critical role in cell fate transitions, development, and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS, including a splicing program that is unique to transgene-independent induced pluripotent stem cells (iPSCs). Changes in the expression of AS factors Zcchc24, Esrp1, Mbnl1/2, and Rbm47 were demonstrated to contribute to phase-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA-binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1. These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency.

The esophageal lumen Is lined by a stratified squamous epithelium comprised of proliferative basal cells that differentiate while migrating toward the luminal surface and eventually desquamate. Rapid epithelial renewal occurs, but the specific cell of origin that supports this high proliferative demand remains unknown. Herein, we have described a long-lived progenitor cell population in the mouse esophageal epithelium that is characterized by expression of keratin 15 (Krt15). Genetic in vivo lineage tracing revealed that the Krt15 promoter marks a long-lived basal cell population able to self-renew, proliferate, and generate differentiated cells, consistent with a progenitor/stem cell population. Transcriptional profiling demonstrated that [Krt15.sup.+] basal cells are molecularly distinct from [Krt15.sup.-] basal cells. Depletion of Krt15-derived cells resulted in decreased proliferation, thereby leading to atrophy of the esophageal epithelium. Further, [Krt15.sup.+] cells were radioresistant and contributed to esophageal epithelial regeneration following radiation-induced injury. These results establish the presence of a long-lived and indispensable [Krt15.sup.+] progenitor cell population that provides additional perspective on esophageal epithelial biology and the widely prevalent diseases that afflict this epithelium., Introduction Adult tissues undergo cellular renewal at variable rates. In some tissues, such as blood, skin, and intestine, stem cells are the source of new cells. In other tissues, such [...]

Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Leukemia cells exhibit a dysregulated developmental program as the result of genetic and epigenetic [...]

RNA-binding proteins of the Musashi (Msi) family are expressed in stem cell compartments and in aggressive tumors, but they have not yet been widely explored in the blood. Here we demonstrate that Msi2 is the predominant form expressed in hematopoietic stem cells (HSCs), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of human MSI2 in a mouse model increases HSC cell cycle progression and cooperates with the chronic myeloid leukemia-associated BCR-ABL1 oncoprotein to induce an aggressive leukemia. MSI2 is overexpressed in human myeloid leukemia cell lines, and its depletion leads to decreased proliferation and increased apoptosis. Expression levels in human myeloid leukemia directly correlate with decreased survival in patients with the disease, thereby defining MSI2 expression as a new prognostic marker and as a new target for therapy in acute myeloid leukemia (AML)., Musashi family RNA-binding proteins have been linked to tissue stem cells but not yet studied extensively in the blood (1,2). Deregulation of key genes that control self-renewal and cell fate [...]

Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be achieved by overexpression of Oct4, Sox2, KIf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming by these transcription factors is a continuous stochastic process where almost all mouse donor cells eventually give rise to WS cells on continued growth and transcription factor expression. Additional inhibition of the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPS cell formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell-division-rate-independent manner. Quantitative analyses define distinct cell-division-rate-dependent and -independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency., Quantifying the efficiency and timescales of crucial events occurring during in vitro reprogramming to pluripotency (1-5) has been problematic due to the cellular and genetic heterogeneity of de novo infected [...]