CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities
Hierarchy and Plasticity in the Intestinal Stem Cell Compartment.
Trends in cell biology
2017; 27 (10): 753?64
Msi RNA-binding proteins control reserve intestinal stem cell quiescence
JOURNAL OF CELL BIOLOGY
2016; 215 (3): 401-413
Somatic stem cells maintain tissue homeostasis by organizing themselves in such a way that they can maintain proliferative output while simultaneously protecting themselves from DNA damage that may lead to oncogenic transformation. There is considerable debate about how such stem cell compartments are organized. Burgeoning evidence from the small intestine and colon provides support for a two-stem cell model involving an actively proliferating but injury-sensitive stem cell and a rare, injury-resistant pool of quiescent stem cells. Parallel with this evidence, recent studies have revealed considerable plasticity within the intestinal stem cell (ISC) compartment. We discuss the evidence for plasticity and hierarchy within the ISC compartment and how these properties govern tissue regeneration and contribute to oncogenic transformation leading to colorectal cancers.
View details for DOI 10.1016/j.tcb.2017.06.006
View details for PubMedID 28732600
View details for PubMedCentralID PMC5612891
Heterogeneity in Readouts of Canonical Wnt Pathway Activity Within Intestinal Crypts
2016; 245 (8): 822-833
Regeneration of the intestinal epithelium is driven by multiple intestinal stem cell (ISC) types, including an active, radiosensitive Wnt(high) ISC that fuels turnover during homeostasis and a reserve, radioresistant Wnt(low/off) ISC capable of generating active Wnt(high) ISCs. We examined the role of the Msi family of oncoproteins in the ISC compartment. We demonstrated that Msi proteins are dispensable for normal homeostasis and self-renewal of the active ISC, despite their being highly expressed in these cells. In contrast, Msi proteins are required specifically for activation of reserve ISCs, where Msi activity is both necessary and sufficient to drive exit from quiescence and entry into the cell cycle. Ablation of Msi activity in reserve ISCs rendered the epithelium unable to regenerate in response to injury that ablates the active stem cell compartment. These findings delineate a molecular mechanism governing reserve ISC quiescence and demonstrate a necessity for the activity of this rare stem cell population in intestinal regeneration.
View details for DOI 10.1083/jcb.201604119
View details for Web of Science ID 000387636900014
View details for PubMedID 27799368
View details for PubMedCentralID PMC5100293
The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins
2015; 13 (11): 2440-2455
Canonical Wnt pathway signaling is necessary for maintaining the proliferative capacity of mammalian intestinal crypt base columnar stem cells (CBCs). Furthermore, dysregulation of the Wnt pathway is a major contributor to disease, including oncogenic transformation of the intestinal epithelium. Given the critical importance of this pathway, numerous tools have been used as proxy measures for Wnt pathway activity, yet the relationship between Wnt target gene expression and reporter allele activity within individual cells at the crypt base remains unclear.Here, we describe a novel Axin2-CreERT2-tdTomato allele that efficiently marks both Wnt(High) CBCs and radioresistant reserve intestinal stem cells. We analyze the molecular and functional identity of Axin2-CreERT2-tdTomato-marked cells using single cell gene expression profiling and tissue regeneration assays and find that Axin2 reporter activity does not necessarily correlate with expression of Wnt target genes and, furthermore, that Wnt target genes themselves vary in their expression patterns at the crypt base.Wnt target genes and reporter alleles can vary greatly in their cell-type specificity, demonstrating that these proxies cannot be used interchangeably. Furthermore, Axin2-CreERT2-tdTomato is a robust marker of both active and reserve intestinal stem cells and is thus useful for understanding the intestinal stem cell compartment. Developmental Dynamics 245:822-833, 2016. © 2016 Wiley Periodicals, Inc.
View details for DOI 10.1002/DVDY.24423
View details for Web of Science ID 000384751600001
View details for PubMedID 27264700
View details for PubMedCentralID PMC4946997
Transformation of the intestinal epithelium by the MSI2 RNA-binding protein
Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.
View details for DOI 10.1016/j.celrep.2015.11.022
View details for Web of Science ID 000367101400014
View details for PubMedID 26673327
View details for PubMedCentralID PMC4894540
Single-Cell Analysis of Proxy Reporter Allele-Marked Epithelial Cells Establishes Intestinal Stem Cell Hierarchy
STEM CELL REPORTS
2014; 3 (5): 876-891
The MSI2 RNA-binding protein is a potent oncogene playing key roles in haematopoietic stem cell homeostasis and malignant haematopoiesis. Here we demonstrate that MSI2 is expressed in the intestinal stem cell compartment, that its expression is elevated in colorectal adenocarcinomas, and that MSI2 loss-of-function abrogates colorectal cancer cell growth. MSI2 gain-of-function in the intestinal epithelium in a drug-inducible mouse model is sufficient to phenocopy many of the morphological and molecular consequences of acute loss of the APC tumour suppressor in the intestinal epithelium in a Wnt-independent manner. Transcriptome-wide RNA-binding analysis indicates that MSI2 acts as a pleiotropic inhibitor of known intestinal tumour suppressors including Lrig1, Bmpr1a, Cdkn1a and Pten. Finally, we demonstrate that inhibition of the PDK-AKT-mTORC1 axis rescues oncogenic consequences of MSI2 induction. Taken together, our findings identify MSI2 as a central component in an unappreciated oncogenic pathway promoting intestinal transformation.
View details for DOI 10.1038/ncomms7517
View details for Web of Science ID 000352720000007
View details for PubMedID 25774828
View details for PubMedCentralID PMC4643281
De Novo Formation of Insulin-Producing "Neo-beta Cell Islets" from Intestinal Crypts
2014; 6 (6): 1046-1058
The recent development of targeted murine reporter alleles as proxies for intestinal stem cell activity has led to significant advances in our understanding of somatic stem cell hierarchies and dynamics. Analysis of these reporters has led to a model in which an indispensable reserve stem cell at the top of the hierarchy (marked by Bmi1 and Hopx reporters) gives rise to active intestinal stem cells (marked by an Lgr5 reporter). Despite these advances, controversy exists regarding the specificity and fidelity with which these alleles distinguish intestinal stem cell populations. Here, we undertake a comprehensive comparison of widely used proxy reporters including both CreERT2 and EGFP cassettes targeted to the Lgr5, Bmi1, and Hopx loci. Single-cell transcriptional profiling of these populations and their progeny reveals that reserve and active intestinal stem cells are molecularly and functionally distinct, supporting a two-stem-cell model for intestinal self-renewal.
View details for DOI 10.1016/j.stemcr.2014.09.011
View details for Web of Science ID 000345118600016
View details for PubMedID 25418730
View details for PubMedCentralID PMC4235148
Embryonic Stem Cell Interactomics: The Beginning of a Long Road to Biological Function
STEM CELL REVIEWS AND REPORTS
2012; 8 (4): 1138-1154
The ability to interconvert terminally differentiated cells could serve as a powerful tool for cell-based treatment of degenerative diseases, including diabetes mellitus. To determine which, if any, adult tissues are competent to activate an islet ? cell program, we performed an in vivo screen by expressing three ? cell "reprogramming factors" in a wide spectrum of tissues. We report that transient intestinal expression of these factors-Pdx1, MafA, and Ngn3 (PMN)-promotes rapid conversion of intestinal crypt cells into endocrine cells, which coalesce into "neoislets" below the crypt base. Neoislet cells express insulin and show ultrastructural features of ? cells. Importantly, intestinal neoislets are glucose-responsive and able to ameliorate hyperglycemia in diabetic mice. Moreover, PMN expression in human intestinal "organoids" stimulates the conversion of intestinal epithelial cells into ?-like cells. Our results thus demonstrate that the intestine is an accessible and abundant source of functional insulin-producing cells.
View details for DOI 10.1016/j.celrep.2014.02.013
View details for Web of Science ID 000333465000010
View details for PubMedID 24613355
View details for PubMedCentralID PMC4245054
Protocol for expansion of undifferentiated human embryonic and pluripotent stem cells in suspension.
Methods in molecular biology (Clifton, N.J.)
2012; 873: 217-226
Embryonic stem cells (ESCs) are capable of unlimited self-renewal while maintaining pluripotency. They are of great interest in regenerative medicine due to their ability to differentiate into all cell types of the three embryonic germ layers. Recently, induced pluripotent stem cells (iPSCs) have shown similarities to ESCs and thus promise great therapeutic potential in regenerative medicine. Despite progress in stem cell biology, our understanding of the exact mechanisms by which pluripotency and self-renewal are established and maintained is largely unknown. A better understanding of these processes may lead to discovery of alternative ways for reprogramming, differentiation and more reliable applications of stem cells in therapies. It has become evident that proteins generally function as members of large complexes that are part of a more complex network. Therefore, the identification of protein-protein interactions (PPI) is an efficient strategy for understanding protein function and regulation. Systematic genome-wide and pathway-specific PPI analysis of ESCs has generated a network of ESC proteins, including major transcription factors. These PPI networks of ESCs may contribute to a mechanistic understanding of self-renewal and pluripotency. In this review we describe different experimental approaches for the identification of PPIs along with various databases. We discuss biological findings and technical challenges encountered with interactome studies of pluripotent stem cells, and provide insight into how interactomics is likely to develop.
View details for DOI 10.1007/s12015-012-9400-9
View details for Web of Science ID 000311510600012
View details for PubMedID 22847281
Long-Term Maintenance of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells in Suspension
STEM CELLS AND DEVELOPMENT
2011; 20 (11): 1911-1923
Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) offer a platform technology with the potential for developmental biology and cell-based therapy. Therefore, robust and cost-effective ways for mass production of them is necessary. Here, we have presented a protocol to grow pluripotent hESCs and hiPSCs in suspension by using a simple, inexpensive, microcarrier-free method. Under this condition, the cells maintained stability during freeze/thaw cycles without the loss of pluripotency markers for extended periods (>1 year). The cells maintained a stable karyotype and showed very similar expression profiles when compared to the adherent culture. The combination of this system with a bioreactor culture system will allow scale up culture of hESCs and hiPSCs needed for clinical and translational applications.
View details for DOI 10.1007/978-1-61779-794-1_13
View details for PubMedID 22528357
Traditionally, undifferentiated pluripotent human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) have been expanded as monolayer colonies in adhesion culture, both in the presence or absence of feeder cells. However, the use of pluripotent stem cells poses the need to scale-up current culture methods. Herein, we present the cultivation of 2 hESC lines (Royan H5 and Royan H6) and 2 hiPSC lines (hiPSC1 and hiPSC4) as carrier-free suspension aggregates for an extended period of time. The cells proliferated over multiple passages kept a stable karyotype, which successfully maintained an undifferentiated state and pluripotency, as determined by marker expressions in addition to in vitro spontaneous and directed differentiation. Additionally, these cells can be easily frozen and thawed without losing their proliferation, karyotype stability, and developmental potential. Transcriptome analysis of the 3 lines revealed that the adherent culture condition was nearly identical to the suspension culture in Royan H5 and hiPSC1, but not in Royan H6. It remains unclear whether this observation at the transcript level is biologically significant. In comparison with recent reports, our study presents a low-cost procedure for long-term suspension expansion of hESCs and hiPSCs with the capability of freeze/thawing, karyotype stability, and pluripotency. Our results will pave the way for scaled up expansion and controlled differentiation of hESCs and hiPSCs needed for cell therapy, research, and industrial applications in a bioreactor culture system.
View details for DOI 10.1089/scd.2010.0517
View details for Web of Science ID 000296587400008
View details for PubMedID 21198400