Maintaining Stem Cell Quiescence Research Articles

NFATc1 Balances Quiescence and Proliferation of Skin Stem Cells

Quiescent adult stem cells reside in specialized niches where they become activated to proliferate and differentiate during tissue homeostasis and injury. How stem cell quiescence is governed is poorly understood. We report here that NFATc1 is preferentially expressed by hair follicle stem cells in their niche, where its expression is activated by BMP signaling upstream and it acts downstream to transcriptionally repress CDK4 and maintain stem cell quiescence. As stem cells become activated during hair growth, NFATc1 is downregulated, relieving CDK4 repression and activating proliferation. When calcineurin/NFATc1 signaling is suppressed, pharmacologically or via complete or conditional NFATc1 gene ablation, stem cells are activated prematurely, resulting in precocious follicular growth. Our findings may explain why patients receiving cyclosporine A for immunosuppressive therapy display excessive hair growth, and unveil a functional role for calcium-NFATc1-CDK4 circuitry in governing stem cell quiescence.

Regulation of Hematopoietic Stem Cell Quiescence - A Novel Role for p53.

Although the p53 tumor suppressor can elicit cell-cycle arrest or apoptosis in hematopoietic cells upon DNA damage, its function during steady-state hematopoiesis is largely unknown. We demonstrated that the Ets transcription factor MEF/ELF4 regulates both HSC proliferation/self-renewal and quiescence, as Mef null mice exhibit greater numbers of hematopoietic stem cells and the Mef null HSCs are more quiescent than normal. As such, the hematopoietic compartment of Mef null mice shows significant resistance to chemotherapy and radiation (Lacorazza et al., Cancer Cell, 2006). In this study, we have investigated the mechanisms utilized by MEF/ELF4 to regulate the quiescence and self-renewal of hematopoietic stem cells, identifying p53 as a key regulator. We have recently found that Mef null mouse embryonic fibroblasts (mefs) accumulate p53 and undergo premature senescence; MEF appears to surpress the expression of p53 by directly upregulating Mdm2 (G. Sashida et al., submitted). We hypothesized that p53 may play a role in the enhanced stem cell quiescence or the increased HSC frequency seen in Mef null mice. To examine this, we generated p53−/− Mef −/− mice and compared HSC biology in the double knock out mice (p53−/− Mef −/−) vs p53 null mice, Mef null mice and wt mice. Loss of p53 decreased the fraction of Pyronin Ylow Lin-Sca-1+ cells, suggesting fewer quiescent HSCs, and staining of CD34-LSK cells for the proliferation marker Ki67 also showed enhanced HSC proliferation in the absence of p53 (with fewer quiescent cells present). These data suggest that p53 promotes quiescence in HSCs, and in the absence of p53, HSCs more readily enter the cell cycle. When we analyzed the DKO (p53−/− Mef −/−) mice, we observed that the percentage of G0 HSCs returned to normal, indicating that p53 is essential for maintaining the enhanced quiescence of MEF null HSCs. p21 is a major target gene of p53 in cells, and has been shown to play an important role in maintaining HSC quiescence. As expeceted, we found elevated levels of p21 mRNA in MEF null LSK cells and reasoned that p21 may account for their enhanced quiescence. We generated p21 −/− Mef −/− mice, which are viable, born at normal mendelian frequency and appear grossly normal. However, cell cycle analysis of HSCs obtained from p21 −/− Mef −/− mice showed that the enhanced quiescence in MEF null HSCs did not depend on p21, indicating that other p53 target genes play an important role in maintaining stem cell quiescence. We therefore utilized transcript profiling (Microarray studies and quantitative PCR analysis) to identify potential p53-regulated genes that may be differentialy expressed in LSK cells isolated from wild type, p53−/−, Mef −/−, and p53−/− Mef −/− mice. By ChiP and luciferase reporter assays, we show for the first time that Gfi-1 and Necdin are direct transcriptional targets of p53 in HSCs and both Gfi-1 and Necdin regulate the enhanced quiescence exhibited in MEF null HSCs. Taken together, our work defines a novel role for p53 in the maintenance of HSC quiescence. Furthermore, HSC quiescence and self-renewal appear to be mediated by different p53 target genes during steady state hematopoiesis.

A Comprehensive Genomic Approach Using Gain of Function and Loss of Function Cell Models and ChIP-on-Chip Technology Identifies Novel Promyelocytic Zinc Finger Protein Target Genes.

The t(11;17)(q23;q21) form of APL involves the production of reciprocal fusion proteins, PLZF-RARα and RARα-PLZF, which mediate malignant transformation by binding to and dysregulating RARα and PLZF target genes. PLZF is expressed in hematopoietic stem cells and is downregulated as cells differentiate. The identification of PLZF target genes including cyclin A2 and MYC is consistent with the hypothesis that PLZF maintains stem cell quiescence by repressing cell cycle driving genes and provides insight into transcriptional pathways disrupted in leukemogenesis. In order to identify additional target genes of PLZF, we constructed a loss of function model in which we suppressed endogenous expression of PLZF using siRNA in KG1a leukemia cells. Our gain of function model consisted of the ectopic expression of PLZF in U937 leukemia cells which do not naturally express PLZF. Expression profiling using GeneChip™ Human Genome U133 Plus 2.0 arrays, which analyze the expression of more than 47,000 transcripts, was performed using both systems. Of the 346 genes identified in the loss of function model, 25% were also regulated by PLZF in the gain of function U937 cell line. Changes in expression of these genes could be direct (through PLZF) or indirect (through secondary effects). In order to determine which genes modulated by changes in PLZF expression are direct transcriptional targets, we immunoprecipitated chromatin using PLZF antibodies in KG1a cells, amplified the products by ligation-mediated PCR and co-hybridized these products with input chromatin to NimbleGen 1.5kB promoter arrays, which represent 24,275 human promoters. Genes bound by PLZF were identified by determining whether consecutively tiled probes were enriched in PLZF-precipitated chromatin as compared to chromatin precipitated with a non-specific antibody. Using a statistical algorithm designed to exclude those probes whose signals of PLZF enrichment might be spuriously identified, we identified 52 genes of the 24,275 on the array as potential PLZF target genes. Strikingly, correlation of these genes with expression analyses revealed that 44% of genes were also significantly regulated by PLZF in the gain of function model and 11% of genes were regulated in the loss of function model. Promoter analyses of a subset of these genes that were identified by ChIP-on-Chip and differentially expressed at least >1.3 fold in PLZF arrays (p<0.05), revealed the presence of a consensus PLZF binding site GTC(C/A)AG in 75% of genes. Analysis of gene ontology for those genes identified by ChIP-on Chip, revealed an enrichment of genes involved in RNA binding and processing as well as genes encoding small G proteins. One gene in particular, RECQL, was directly bound by PLZF in the ChIP-on-Chip assay and transcriptionally regulated by PLZF in both KG1a loss of function and U937 gain of function models. The RECQL protein is a member of the RecQ family of DNA helicases, a class of genes whose mutation is associated with genomic instability tumorigenesis and premature ageing. These data indicate a robust system for the identification of PLZF targets and suggest that PLZF may play a role in genome integrity.

An enteroendocrine cell‐based model for a quiescent intestinal stem cell niche

We have shown that the kinetics of conversion of intestinal crypt cell populations to a partially or wholly mutant phenotype are consistent with a model in which each crypt contains an infrequently dividing 'deep' stem cell that is the progenitor of several more frequently dividing 'proximate' stem cells. An assumption of our model is that each deep stem cell exists in a growth inhibitory niche. We have used information from the literature to develop a model for a quiescent intestinal stem cell niche. This niche is postulated to be primarily defined by an enteroendocrine cell type that maintains stem cell quiescence by secretion of growth inhibitory peptides such as somatostatin and guanylin/uroguanylin. Consistent with this model, there is evidence that the proteins postulated as defining a growth-inhibitory stem cell niche can act as intestinal tumour suppressors. Confirmation that a growth-inhibitory niche does exist would have important implications for our understanding of intestinal homeostasis and tumorigenesis.

Ex vivo targeting of p21Cip1/Waf1permits relative expansion of human hematopoietic stem cells

AbstractRelative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling, we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor, p21Cip1/Waf1(p21), have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling, we tested whether reduction of p21 alone in human adult–derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control, manipulated cells. Further, we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore, lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion.