David T. Breault

Defining Molecular Cornerstones during Fibroblast to iPS Cell Reprogramming in Mouse

Ectopic expression of the transcription factors Oct4, Sox2, c-Myc, and Klf4 in fibroblasts generates induced pluripotent stem (iPS) cells. Little is known about the nature and sequence of molecular events accompanying nuclear reprogramming. Using doxycycline-inducible vectors, we have shown that exogenous factors are required for about 10 days, after which cells enter a self-sustaining pluripotent state. We have identified markers that define cell populations prior to and during this transition period. While downregulation of Thy1 and subsequent upregulation of SSEA-1 occur at early time points, reactivation of endogenous Oct4, Sox2, telomerase, and the silent X chromosome mark late events in the reprogramming process. Cell sorting with these markers allows for a significant enrichment of cells with the potential to become iPS cells. Our results suggest that factor-induced reprogramming is a gradual process with defined intermediate cell populations that contain the majority of cells poised to become iPS cells.

Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells

The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5+) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5+ cells. mTert-expressing cells distribute in a pattern along the crypt–villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert+ cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.

Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells

Stem cells hold great promise for regenerative medicine, but remain elusive in many tissues in part because universal markers of “stemness” have not been identified. The ribonucleoprotein complex telomerase catalyzes the extension of chromosome ends, and its expression is associated with failure of cells to undergo cellular senescence. Because such resistance to senescence is a common characteristic of many stem cells, we hypothesized that telomerase expression may provide a selective biomarker for stem cells in multiple tissues. In fact, telomerase expression has been demonstrated within hematopoietic stem cells. We therefore generated mouse telomerase reverse transcriptase (mTert)-GFP-transgenic mice and assayed the ability of mTert-driven GFP to mark tissue stem cells in testis, bone marrow (BM), and intestine. mTert-GFP mice were generated by using a two-step embryonic stem cell-based strategy, which enabled primary and secondary screening of stably transfected clones before blastocyst injection, greatly increasing the probability of obtaining mTert reporter mice with physiologically appropriate regulation of GFP expression. Analysis of adult mice showed that GFP is expressed in differentiating male germ cells, is enriched among BM-derived hematopoietic stem cells, and specifically marks long-term BrdU-retaining intestinal crypt cells. In addition, telomerase-expressing GFP+ BM cells showed long-term, serial, multilineage BM reconstitution, fulfilling the functional definition of hematopoietic stem cells. Together, these data provide direct evidence that mTert-GFP expression marks progenitor cells in blood and small intestine, validating these mice as a useful tool for the prospective identification, isolation, and functional characterization of progenitor/stem cells from multiple tissues.

Small intestinal stem cell markers

Stem cells hold great promise for regenerative medicine but remain elusive in many tissues, including the small intestine, where it is well accepted that the epithelium is maintained by intestinal stem cells located in the crypts. The lack of established markers to prospectively identify intestinal stem cells has necessitated the use of indirect analysis, e.g. long‐term label retention, which is based on the hypothesis that intestinal stem cells are slow‐cycling. Several intestinal stem cell markers have been proposed, including Musashi‐1, BMPR1α, phospho‐PTEN, DCAMKL1, Eph receptors and integrins, but their validity, using functional and/or lineage tracing assays, has yet to be confirmed. Recently, Lgr5 has been identified by lineage tracing as an intestinal stem cell marker. In this review we summarize what is known about the currently reported intestinal stem cell markers and provide a rationale for developing model systems whereby intestinal stem cells can be functionally validated.

Adrenocortical Zonation Results from Lineage Conversion of Differentiated Zona Glomerulosa Cells

Lineage conversion of differentiated cells in response to hormonal feedback has yet to be described. To investigate this, we studied the adrenal cortex, which is composed of functionally distinct concentric layers that develop postnatally, the outer zona glomerulosa (zG) and the inner zona fasciculata (zF). These layers have separate functions, are continuously renewed in response to physiological demands, and are regulated by discrete hormonal feedback loops. Their cellular origin, lineage relationship, and renewal mechanism, however, remain poorly understood. Cell-fate mapping and gene-deletion studies using zG-specific Cre expression demonstrate that differentiated zG cells undergo lineage conversion into zF cells. In addition, zG maintenance is dependent on the master transcriptional regulator Steroidogenic Factor 1 (SF-1), and zG-specific Sf-1 deletion prevents lineage conversion. These findings demonstrate that adrenocortical zonation and regeneration result from lineage conversion and may provide a paradigm for homeostatic cellular renewal in other tissues.

Frizzled proteins are colonic epithelial receptors for C. difficile toxin B

Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR–Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity towards FZD1, 2 and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signalling. FZD1/2/7 triple-knockout cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7-knockout mice, combined with knockdown of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7-knockout mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.

Reprogrammed Stomach Tissue as a Renewable Source of Functional β Cells for Blood Glucose Regulation

The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin(+) cells after undergoing cellular reprogramming. Here, we show that cells of the antral stomach have a previously unappreciated propensity for conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic β cells, and expression of β cell reprogramming factors in vivo converts antral cells efficiently into insulin(+) cells with close molecular and functional similarity to β cells. Induced GI insulin(+) cells can suppress hyperglycemia in a diabetic mouse model for at least 6 months and regenerate rapidly after ablation. Reprogramming of antral stomach cells assembled into bioengineered mini-organs in vitro yielded transplantable units that also suppressed hyperglycemia in diabetic mice, highlighting the potential for development of engineered stomach tissues as a renewable source of functional β cells for glycemic control.

Regulation of Gene Expression in the Intestinal Epithelium

Regulation of gene expression within the intestinal epithelium is complex and controlled by various signaling pathways that regulate the balance between proliferation and differentiation. Proliferation is required both to grow and to replace cells lost through apoptosis and attrition, yet in all but a few cells, differentiation must take place to prevent uncontrolled growth (cancer) and to provide essential functions. In this chapter, we review the major signaling pathways underlying regulation of gene expression within the intestinal epithelium, based primarily on data from mouse models, as well as specific morphogens and transcription factor families that have a major role in regulating intestinal gene expression, including the Hedgehog family, Forkhead Box (FOX) factors, Homeobox (HOX) genes, ParaHox genes, GATA transcription factors, canonical Wnt/β-catenin signaling, EPH/Ephrins, Sox9, BMP signaling, PTEN/PI3K, LKB1, K-RAS, Notch pathway, HNF, and MATH1. We also briefly highlight important emerging areas of gene regulation, including microRNA (miRNA) and epigenetic regulation.

Dormant Intestinal Stem Cells Are Regulated by PTEN and Nutritional Status

The cellular and molecular mechanisms underlying adaptive changes to physiological stress within the intestinal epithelium remain poorly understood. Here, we show that PTEN, a negative regulator of the PI3K→AKT→mTORC1-signaling pathway, is an important regulator of dormant intestinal stem cells (d-ISCs). Acute nutrient deprivation leads to transient PTEN phosphorylation within d-ISCs and a corresponding increase in their number. This release of PTEN inhibition renders d-ISCs functionally poised to contribute to the regenerative response during re-feeding via cell-autonomous activation of the PI3K→AKT→mTORC1 pathway. Consistent with its role in mediating cell survival, PTEN is required for d-ISC maintenance at baseline, and intestines lacking PTEN have diminished regenerative capacity after irradiation. Our results highlight a PTEN-dependent mechanism for d-ISC maintenance and further demonstrate the role of d-ISCs in the intestinal response to stress.

Tales From the Crypt: The Expanding Role of Slow Cycling Intestinal Stem Cells

Similar to other highly self-renewing tissues, the intestinal epithelium contains both slowly and rapidly cycling progenitor/stem cells, though their relationship has been largely unexplored. Two recent reports in Nature (Tian et al., 2011) and Science (Takeda et al., 2011) shed new light on their dynamic interplay.