The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells.

The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity.

Altered leukotriene B4 metabolism in CYP4F18-deficient mice does not impact inflammation following renal ischemia.

Inflammatory responses to infection and injury must be restrained and negatively regulated to minimize damage to host tissue. One proposed mechanism involves enzymatic inactivation of the pro-inflammatory mediator leukotriene B4, but it is difficult to dissect the roles of various metabolic enzymes and pathways. A primary candidate for a regulatory pathway is omega oxidation of leukotriene B4 in neutrophils, presumptively by CYP4F3A in humans and CYP4F18 in mice. This pathway generates ω, ω-1, and ω-2 hydroxylated products of leukotriene B4, depending on species. We created mouse models targeting exons 8 and 9 of the Cyp4f18 allele that allows both conventional and conditional knockouts of Cyp4f18. Neutrophils from wild-type mice convert leukotriene B4 to 19-hydroxy leukotriene B4, and to a lesser extent 18-hydroxy leukotriene B4, whereas these products were not detected in neutrophils from conventional Cyp4f18 knockouts. A mouse model of renal ischemia-reperfusion injury was used to investigate the consequences of loss of CYP4F18 in vivo. There were no significant changes in infiltration of neutrophils and other leukocytes into kidney tissue as determined by flow cytometry and immunohistochemistry, or renal injury as assessed by histological scoring and measurement of blood urea nitrogen. It is concluded that CYP4F18 is necessary for omega oxidation of leukotriene B4 in neutrophils, and is not compensated by other CYP enzymes, but loss of this metabolic pathway is not sufficient to impact inflammation and injury following renal ischemia-reperfusion in mice.

Genome-wide map of nuclear protein degradation shows NCoR1 turnover as a key to mitochondrial gene regulation.

Transcription factor activity and turnover are functionally linked, but the global patterns by which DNA-bound regulators are eliminated remain poorly understood. We established an assay to define the chromosomal location of DNA-associated proteins that are slated for degradation by the ubiquitin-proteasome system. The genome-wide map described here ties proteolysis in mammalian cells to active enhancers and to promoters of specific gene families. Nuclear-encoded mitochondrial genes in particular correlate with protein elimination, which positively affects their transcription. We show that the nuclear receptor corepressor NCoR1 is a key target of proteolysis and physically interacts with the transcription factor CREB. Proteasome inhibition stabilizes NCoR1 in a site-specific manner and restrains mitochondrial activity by repressing CREB-sensitive genes. In conclusion, this functional map of nuclear proteolysis links chromatin architecture with local protein stability and identifies proteolytic derepression as highly dynamic in regulating the transcription of genes involved in energy metabolism.

Differential regulation of myeloid leukemias by the bone marrow microenvironment.

Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM) and may be the cause of relapse following chemotherapy. Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. CD44 (refs. 3,4) and interleukin-6 (ref. 5) have been implicated previously in the LSC niche. Transforming growth factor-ß1 (TGF-ß1) is released during bone remodeling and plays a part in maintenance of CML LSCs, but a role for TGF-ß1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor attenuates BCR-ABL1 oncogene-induced CML-like myeloproliferative neoplasia (MPN) but enhances MLL-AF9 oncogene-induced AML in mouse transplantation models, possibly through opposing effects of increased TGF-ß1 on the respective LSCs. PTH treatment caused a 15-fold decrease in LSCs in wild-type mice with CML-like MPN and reduced engraftment of immune-deficient mice with primary human CML cells. These results demonstrate that LSC niches in CML and AML are distinct and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSCs, a prerequisite for the cure of CML.

Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance.

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad-) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad- cells. Similarly, proliferation of the 3+Ecad- cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3-Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad- subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad- cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics.

Densely interconnected transcriptional circuits control cell states in human hematopoiesis.

Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.

Mixed chimerism, lymphocyte recovery, and evidence for early donor-specific unresponsiveness in patients receiving combined kidney and bone marrow transplantation to induce tolerance.

BACKGROUND: We have previously reported operational tolerance in patients receiving human leukocyte antigen-mismatched combined kidney and bone marrow transplantation (CKBMT). We now report on transient multilineage hematopoietic chimerism and lymphocyte recovery in five patients receiving a modified CKBMT protocol and evidence for early donor-specific unresponsiveness in one of these patients. METHODS: Five patients with end-stage renal disease received CKBMT from human leukocyte antigen-mismatched, haploidentical living-related donors after modified nonmyeloablative conditioning. Polychromatic flow cytometry was used to assess multilineage chimerism and lymphocyte recovery posttransplant. Limiting dilution analysis was used to assess helper T-lymphocyte reactivity to donor antigens. RESULTS: Transient multilineage mixed chimerism was observed in all patients, but chimerism became undetectable by 2 weeks post-CKBMT. A marked decrease in T- and B-lymphocyte counts immediately after transplant was followed by gradual recovery. Initially, recovering T cells were depleted of CD45RA+/CD45RO(-) "naïve-like" cells, which have shown strong recovery in two patients, and CD4:CD8 ratios increased immediately after transplant but then declined markedly. Natural killer cells were enriched in the peripheral blood of all patients after transplant.For subject 2, a pretransplant limiting dilution assay revealed T helper cells recognizing both donor and third-party peripheral blood mononuclear cells. However, the antidonor response was undetectable by day 24, whereas third-party reactivity persisted. CONCLUSION: These results characterize the transient multilineage mixed hematopoietic chimerism and recovery of lymphocyte subsets in patients receiving a modified CKBMT protocol. The observations are relevant to the mechanisms of donor-specific tolerance in this patient group.

Mullerian inhibiting substance preferentially inhibits stem/progenitors in human ovarian cancer cell lines compared with chemotherapeutics.

Cancer stem cells are proposed to be tumor-initiating cells capable of tumorigenesis, recurrence, metastasis, and drug resistance, and, like somatic stem cells, are thought to be capable of unlimited self-renewal and, when stimulated, proliferation and differentiation. Here we select cells by expression of a panel of markers to enrich for a population with stem cell-like characteristics. A panel of eight was initially selected from 95 human cell surface antigens as each was shared among human ovarian primary cancers, ovarian cancer cell lines, and normal fimbria. A total of 150 combinations of markers were reduced to a panel of three--CD44, CD24, and Epcam--which selected, in three ovarian cancer cell lines, those cells which best formed colonies. Cells expressing CD44, CD24, and Epcam exhibited stem cell characteristics of shorter tumor-free intervals in vivo after limiting dilution, and enhanced migration in invasion assays in vitro. Also, doxorubicin, cisplatin, and paclitaxel increased this enriched population which, conversely, was significantly inhibited by Müllerian inhibiting substance (MIS) or the MIS mimetic SP600125. These findings demonstrate that flow cytometry can be used to detect a population which shows differential drug sensitivity, and imply that treatment of patients can be individualized to target both stem/progenitor cell enriched and nonenriched subpopulations. The findings also suggest that this population, amenable to isolation by flow cytometry, can be used to screen for novel treatment paradigms, including biologic agents such as MIS, which will improve outcomes for patients with ovarian cancer.

Protein kinase CK1alphaLS promotes vascular cell proliferation and intimal hyperplasia.

Protein kinase CK1alpha regulates several fundamental cellular processes including proliferation and differentiation. Up to four forms of this kinase are expressed in vertebrates resulting from alternative splicing of exons; these exons encode either the L-insert located within the catalytic domain or the S-insert located at the C terminus of the protein. Whereas the L-insert is known to target the kinase to the nucleus, the functional significance of nuclear CK1alphaLS has been unclear. Here we demonstrate that selective L-insert-targeted short hairpin small interfering RNA-mediated knockdown of CK1alphaLS in human vascular endothelial cells and vascular smooth muscle cells impairs proliferation and abolishes hydrogen peroxide-stimulated proliferation of vascular smooth muscle cells, with the cells accumulating in G(0)/G(1). In addition, selective knockdown of CK1alphaLS in cultured human arteries inhibits vascular activation, preventing smooth muscle cell proliferation, intimal hyperplasia, and proteoglycan deposition. Knockdown of CK1alphaLS results in the harmonious down-regulation of its target substrate heterogeneous nuclear ribonucleoprotein C and results in the altered expression or alternative splicing of key genes involved in cellular activation including CXCR4, MMP3, CSF2, and SMURF1. Our results indicate that the nuclear form of CK1alpha in humans, CK1alphaLS, plays a critical role in vascular cell proliferation, cellular activation, and hydrogen peroxide-mediated mitogenic signal transduction.

Uterine leiomyomas exhibit fewer stem/progenitor cell characteristics when compared with corresponding normal myometrium.

Uterine leiomyomas (also known as uterine fibroids) are the most common benign tumors of female reproductive tract and are the single most common indication for hysterectomies. Despite their high prevalence, the exact pathogenesis of these benign tumors is still unknown. One possible mechanism for leiomyoma formation is dysregulation of mesenchymal stem cell activity. Mesenchymal stem cells have been identified in both human and murine uteri and cancer stem cells have been identified in female reproductive malignancies. We compared stem/progenitor cell characteristics in both normal myometrium and the corresponding leiomyoma of patient's undergoing hysterectomies. We found that leiomyoma cells form fewer mesenchymal stem cell colonies and exhibit less Hoechst dye-excluding side population (SP) activity, which is a function associated with progenitor cells in other tissues, than cells isolated from normal myometrium. Whereas in normal myometrium, we observed heterogeneous expression of CD90, a cell surface marker associated the with differentiation potential of uterine fibroblasts, in leiomyomas, we observed homogenous expression of CD90, suggesting leiomyoma cells are more terminally differentiated. Furthermore, we found that while leiomyoma cells could only produce CD90 expressing cells, both CD90+ and CD90- myometrial cells could reestablish their original heterogeneous CD90 profile when expanded in vitro. These results suggest that normal myometrium contains cells with stem/progenitor cell activities that are absent in leiomyomas.

RIAM regulates the cytoskeletal distribution and activation of PLC-gamma1 in T cells.

Rap1-guanosine triphosphate (GTP)-interacting adaptor molecule (RIAM) plays a critical role in actin reorganization and inside-out activation of integrins in lymphocytes and platelets. We investigated the role of RIAM in T cell receptor (TCR)-mediated signaling. Although phosphorylation of the kinase ZAP-70 and formation of a signalosome recruited to the adaptor protein LAT were unaffected, elimination of endogenous RIAM by short hairpin RNA impaired generation of inositol 1,4,5-trisphosphate, mobilization of intracellular calcium ions (Ca(2+)), and translocation of the transcription factor NFAT to the nucleus. The activation of Ras guanine nucleotide-releasing protein 1 was also impaired, which led to the diminished expression of the gene encoding interleukin-2. These events were associated with the impaired translocation of phosphorylated phospholipase C-gamma1 (PLC-gamma1) to the actin cytoskeleton, which was required to bring PLC-gamma1 close to its substrate phosphatidylinositol 4,5-bisphosphate, and were reversed by reconstitution of cells with RIAM. Thus, by regulating the localization of PLC-gamma1, RIAM plays a central role in TCR signaling and the transcription of target genes.

Flow cytometry and the stability of phycoerythrin-tandem dye conjugates.

Routine clinical flow cytometric procedures demand rigorous, simple, and reproducible procedures for spectral compensation. The current, often laborious, spectral compensation procedures are the result of variability in instrument settings, instrument performance, and variability in reagents. In particular, the use of tandem dye conjugates necessitates elaborate spectral compensation procedures that need to be applied frequently. Manufacturer, lot number, and handling procedures are considered the key aspects affecting the fluorescence characteristics of tandem dyes. A better understanding of how specific conditions affect the variability in emission spectra of tandem dyes can lead to a considerable increase in reliability of measurements and a potential simplification of setup procedures for routine, clinical flow cytometry. We investigated the effect of light exposure, handling, and storage conditions on the fluorescence characteristics of some common phycoerythrin tandem fluorochromes. In general, PE-Cy5 showed the lowest degradation rates, whereas PE-Cy7 showed the highest. During storage, long-term degradation rates were lowest for reagents packaged using an extra light protective approach. Under these conditions, a degradation rate of 0.9%/month of a PE-Cy7 conjugate decreased to 0.3%/month. As degradation rates were minimized, we studied the effect of slow degradation of a set of tandem dye conjugates on compensation matrix values over several months. Finally, we explored the effect of slow degradation on flow cytometric analysis using the same compensation settings for extended periods for an analysis template with preset regions and gating strategies.

Dysfunctional expansion of hematopoietic stem cells and block of myeloid differentiation in lethal sepsis.

Severe sepsis is one of the leading causes of death worldwide. High mortality rates in sepsis are frequently associated with neutropenia. Despite the central role of neutrophils in innate immunity, the mechanisms causing neutropenia during sepsis remain elusive. Here, we show that neutropenia is caused in part by apoptosis and is sustained by a block of hematopoietic stem cell (HSC) differentiation. Using a sepsis murine model, we found that the human opportunistic bacterial pathogen Pseudomonas aeruginosa caused neutrophil depletion and expansion of the HSC pool in the bone marrow. "Septic" HSCs were significantly impaired in competitive repopulation assays and defective in generating common myeloid progenitors and granulocyte-monocyte progenitors, resulting in lower rates of myeloid differentiation in vitro and in vivo. Delayed myeloid-neutrophil differentiation was further mapped using a lysozyme-green fluorescent protein (GFP) reporter mouse. Pseudomonas's lipopolysaccharide was necessary and sufficient to induce myelosuppresion and required intact TLR4 signaling. Our results establish a previously unrecognized link between HSC regulation and host response in severe sepsis and demonstrate a novel role for TLR4.

Advances in complex multiparameter flow cytometry technology: Applications in stem cell research.

Flow cytometry and cell sorting are critical tools in stem cell research. Recent advances in flow cytometric hardware, reagents, and software have synergized to permit the stem cell biologist to more fully identify and isolate rare cells based on their immunofluorescent and light scatter characteristics. Some of these improvements include physically smaller air-cooled lasers, new designs in optics, new fluorescent conjugate-excitation pairs, and improved software to visualize data, all which combine to open up new horizons in the study of stem cells, by enhancing the resolution and specificity of inquiry. In this review, these recent improvements in technology will be outlined and important cell surface and functional antigenic markers useful for the study of stem cells described.

Normal ovarian surface epithelial label-retaining cells exhibit stem/progenitor cell characteristics.

Ovulation induces cyclic rupture and regenerative repair of the ovarian coelomic epithelium. This process of repeated disruption and repair accompanied by complex remodeling typifies a somatic stem/progenitor cell-mediated process. Using BrdU incorporation and doxycycline inducible histone2B-green fluorescent protein pulse-chase techniques, we identify a label-retaining cell population in the coelomic epithelium of the adult mouse ovary as candidate somatic stem/progenitor cells. The identified population exhibits quiescence with asymmetric label retention, functional response to estrous cycling in vivo by proliferation, enhanced growth characteristics by in vitro colony formation, and cytoprotective mechanisms by enrichment for the side population. Together, these characteristics identify the label-retaining cell population as a candidate for the putative somatic stem/progenitor cells of the coelomic epithelium of the mouse ovary.

MicroRNA-mediated control of cell fate in megakaryocyte-erythrocyte progenitors.

Lineage specification is a critical issue in developmental and regenerative biology. We hypothesized that microRNAs (miRNAs) are important participants in those processes and used the poorly understood regulation of megakaryocyte-erythrocyte progenitors (MEPs) in hematopoiesis as a model system. We report here that miR-150 modulates lineage fate in MEPs. Using a novel methodology capable of profiling miRNA expression in small numbers of primary cells, we identify miR-150 as preferentially expressed in the megakaryocytic lineage. Through gain- and loss-of-function experiments, we demonstrate that miR-150 drives MEP differentiation toward megakaryocytes at the expense of erythroid cells in vitro and in vivo. Moreover, we identify the transcription factor MYB as a critical target of miR-150 in this regulation. These experiments show that miR-150 regulates MEP fate, and thus establish a role for miRNAs in lineage specification of mammalian multipotent cells.

Bone marrow transplantation generates immature oocytes and rescues long-term fertility in a preclinical mouse model of chemotherapy-induced premature ovarian failure.

PURPOSE: Although early menopause frequently occurs in female cancer patients after chemotherapy (CTx), bone marrow (BM) transplantation (BMT) has been linked to an unexplained return of ovarian function and fertility in some survivors. Studies modeling this in mice have shown that BMT generates donor-derived oocytes in CTx-treated recipients. However, a subsequent report claimed that ovulated eggs are not derived from BM and that BM-derived oocytes reported previously are misidentified immune cells. This study was conducted to further clarify the impact of BMT on female reproductive function after CTx using a preclinical mouse model. METHODS: Female mice were administered CTx followed by BMT using coat color-mismatched female donors. After housing with males, the number of pregnancies and offspring genotype were recorded. For cell tracking, BM from germline-specific green fluorescent protein-transgenic mice was transplanted into CTx-treated wild-type recipients. Immune cells were sorted from blood and analyzed for germline markers. RESULTS: BMT rescued long-term fertility in CTx-treated females, but all offspring were derived from the recipient germline. Cell tracking showed that donor-derived oocytes were generated in ovaries of recipients after BMT, and two lines of evidence dispelled the claim that these oocytes are misidentified immune cells. CONCLUSION: These data from a preclinical mouse model validate a testable clinical strategy for preserving or resurrecting ovarian function and fertility in female cancer patients after CTx, thus aligning with recommendations of the 2005 National Cancer Institute Breast Cancer Progress Review Group and President's Cancer Panel to prioritize research efforts aimed at improving the quality of life in cancer survivors.

Regulatory T-cell recovery in recipients of haploidentical nonmyeloablative hematopoietic cell transplantation with a humanized anti-CD2 mAb, MEDI-507, with or without fludarabine.

OBJECTIVE: We have evaluated T-cell reconstitution and reactivity in patients receiving nonmyeloablative haploidentical hematopoietic cell transplantation (HCT) protocols involving an anti-CD2 monoclonal antibody (MEDI 507) to treat chemorefractory hematopoietic malignancies. METHODS: Three cohorts of four patients each and one cohort of six patients received one of four Medi-507-based regimens, all of which included cyclophosphamide, thymic irradiation, and a short posttransplantation course of cyclosporine. RESULTS: Following marked T-cell depletion, initially recovering CD4 and CD8 T cells were mainly memory-type cells. A high percentage of CD4 T cells expressed high levels of CD25 in recipients of all protocols, except the only protocol to include fludarabine, early post-HCT. CD25 expression varied inversely with T-cell concentrations in blood. CD25(high) CD4 T cells expressed Foxp3 and cytotoxic T-lymphocyte-associated protein 4, indicating that they were regulatory T cells (Treg). CONCLUSIONS: Fludarabine treatment prevents Treg enrichment after haploidentical nonmyeloablative stem cell transplantation, presumably by depleting recipient Tregs. In vitro analyses of allorecognition were consistent with a cytokine-mediated rejection process in one case and in another provided proof of principle that mixed chimerism achieved without graft-vs-host disease induces donor- and recipient-specific tolerance. More reliable achievement of this outcome could provide a promising strategy for organ allograft tolerance induction.

Adult mouse myometrial label-retaining cells divide in response to gonadotropin stimulation.

Conditional deletion of beta-catenin in the Müllerian duct mesenchyme results in a degenerative uterus characterized by replacement of the myometrial smooth muscle with adipose tissue. We hypothesized that the mouse myometrium houses somatic smooth muscle progenitor cells that are hormonally responsive and necessary for remodeling and regeneration during estrous cycling and pregnancy. We surmise that the phenotype observed in beta-catenin conditionally deleted mice is the result of dysregulation of these progenitor cells. The objective of this study was to identify the mouse myometrial smooth muscle progenitor cell and its niche, define the surface marker phenotype, and show a functional response of these cells to normal myometrial cycling. Uteri were labeled with 5-bromo-2'-deoxyuridine (BrdU) and chased for up to 14 weeks. Myometrial label-retaining cells (LRCs) were observed in the myometrium and stroma throughout the chase period. After 12 weeks, phenotypic analysis of the LRCs by immunofluorescence demonstrated that the majority of LRCs colocalized with alpha-smooth muscle actin, estrogen receptor-alpha, and beta-catenin. Flow cytometry of myometrial cells identified a myometrial Hoechst 33342 effluxing "side population" that expresses MISRII-Cre-driven YFP. Functional response of LRCs was investigated by human chorionic gonadotropin stimulation of week 12 chase mice and demonstrated sequential proliferation of LRCs in the endometrial stroma, followed by the myometrium. These results suggest that conventional myometrial regeneration and repair is executed by hormonally responsive stem or progenitor cells derived from the Müllerian duct mesenchyme. Disclosure of potential conflicts of interest is found at the end of this article.

Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.

Stem-cell ageing is thought to contribute to altered tissue maintenance and repair. Older humans experience increased bone marrow failure and poorer haematologic tolerance of cytotoxic injury. Haematopoietic stem cells (HSCs) in older mice have decreased per-cell repopulating activity, self-renewal and homing abilities, myeloid skewing of differentiation, and increased apoptosis with stress. Here we report that the cyclin-dependent kinase inhibitor p16INK4a, the level of which was previously noted to increase in other cell types with age, accumulates and modulates specific age-associated HSC functions. Notably, in the absence of p16INK4a, HSC repopulating defects and apoptosis were mitigated, improving the stress tolerance of cells and the survival of animals in successive transplants, a stem-cell-autonomous tissue regeneration model. Inhibition of p16INK4a may ameliorate the physiological impact of ageing on stem cells and thereby improve injury repair in aged tissue.