Self-renewal Capacity Of Hematopoietic Stem Cells Research Articles

Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation

Hematopoietic stem cells (HSCs) maintain blood production. How often mouse HSCs divide and whether each HSC contributes simultaneously, sequentially, or repetitively to hematopoiesis remains to be determined. We track division of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled HSC in vivo. We found that, in steady-state mice, bone marrow cells capable of reconstituting lifelong hematopoiesis are found within both fast-cycling (undergoing five or more divisions in 7 wk) and quiescent (undergoing zero divisions in 12-14 wk) lineage marker-negative c-Kit(+) Sca-1(+) populations. The contribution of each population to hematopoiesis can fluctuate with time, and cells with extensive proliferative history are prone to return to quiescence. Furthermore, injection of the bacterial component lipopolysaccharide increased the proliferation and self-renewal capacity of HSCs. These findings suggest a model in which all HSCs undergo dynamic and demand-adapted entry into and exit out of the cell cycle over time. This may facilitate a similar degree of turnover of the entire HSC pool at the end of life.

Mdm2 Is Required for Survival of Hematopoietic Stem Cells/Progenitors via Dampening of ROS-Induced p53 Activity

Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2(-/-) p53(515C/515C) mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS and cell cycle arrest, senescence, and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized because of ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.

A Novel Mouse Xenograft Model for Generation of JAK2 V617F Driven Leukemic Stem Cells From Human Embryonic Stem Cells.

Abstract 1433Poster Board I-456 IntroductionMany myeloproliferative neoplasms (MPN) arise in the hematopoietic stem cell (HSC) population upon acquisition of a somatic mutation leading to an aberrantly activated tyrosine kinase, JAK2 V617F. Identification of cell type and context specific genetic and epigenetic events driving leukemic transformation of MPNs has been hampered by the limited supply of the MPN patient blood and marrow samples. Recent publications demonstrate that human embryonic stem cells (hESC) can be differentiated to HSCs efficiently upon coculture with stromal cell lines derived from the murine aorta-gonad-mesonephros (AGM) region. These methods can provide a potentially limitless supply of self-renewing stem cells. We have utilized two hES cell lines, H9 and Hues9 cells, to differentiate toward HSC. We developed novel in vitro and in vivo model systems involving lentivirally enforced expression of genes, such as JAK2V617F, wild type JAK2 and/or activated beta-catenin, in an attempt to generate MPN stem cells. Methods and ResultsTo generate MPN stem cells in initial experiments (n=4), karyotypically normal Hues9 cells were lentivirally transduced with luciferase-GFP and constitutively active beta-catenin (Beta-Cat) alone or in combination with 1) JAK2 V617F, 2) wild-type JAK2, or 3) backbone lentiviral vector. The engraftment capacity of these genetically modified Hues9 cells was compared following intrahepatic transplantation (105-106 cells/mouse) of neonatal RAG2-/-gamma c-/- mice. Lentiviral transduction efficiency was 50-60%. Bioluminescent imaging demonstrated human engraftment at 6 and 11 weeks in all subgroups. The greatest proportion of human cells was noted in the thymus at week 6 and 11, and in the spleens at week 6. There was a 2.4 fold increase in thymic engraftment of CD34+CD45+ cells derived from JAK2 V617F transduced Hues9 cells and a 1.7-fold increase in CD38+CD45+ cells in the JAK2-wild type subset of mice compared with backbone controls. However, overall engraftment rates were low. To determine if hESC differentiation into CD34+ cells improved engraftment, both H9 and Hues9 hES cells were plated in 6 well plates onto mitomycin treated stromal cell lines AM20.1B4 (AM) and UG26.1B6 (UG) that were derived from aorta/mesenchyme and the urogenital ridge of the AGM region of 10 dpc murine embryos respectively. Peak CD34 expression was observed on day 10-12 (n=4) with a sharp decline in CD34 expression by day 18 (n=2). Notably, CD34+ cells began to express CD31 by day 18 with over 50% of CD34+ cells expressing CD31 by day 21 (n=2). Both undifferentiated H9 and Hues9 hES cells as well as CD34+ derivatives expressed CD90. RUNX-1 and GATA-1 expression was higher on day 12 compared to day 18 by Q-RT-PCR in H9 cultured on UG stroma. Plating of 100,000 hESC from day 10-12 cultures with UG stroma generated colonies in hematopoietic progenitor assays with in vitro replating potential while day 18-24 hESC cultures did not. In vitro derived CD34+ cells from day 13 H9 cultures with UG stroma were double selected for CD34 using immunomagnetic beads. The cells were transduced for 6 hr with lentiviral luciferase GFP, Beta-Cat alone (N=4), or Beta-Cat and lentiviral backbone vector (LV) (N=4), wild-type JAK2 (N=4) or JAK2 V617F. The cells were transplanted intrahepatically (5×104 cells/mouse) as above and monitored for engraftment by bioluminescent imaging. Human engraftment was detected at 6 weeks in 2 out of 3 JAK2 V617F/Beta-Cat, 1 of 4 wild-type JAK2/ Beta-Cat, and 0 of 4 Beta-Cat transplanted mice. ConclusionsCo-culturing of hES cell lines, H9 and Hues9, on AGM stromal cell lines appears to generate replatable colonies in hematopoietic progenitor assays. Furthermore, engraftment of lentiviral luciferase transduced hESC in RAG2-/-gamma c-/- mouse hematopoietic organs can be monitored by non-invasive bioluminescent imaging. Although enhanced by lentivirally enforced expression of genes that promote hematopoietic differentiation, hematopoietic engraftment is not robust suggesting a relative dearth of environmental cues that promote human hematopoietic differentiation. Transplantation of genetically engineered hESC together with supportive stroma as well as secondary transplantation will be used to monitor changes in hESC derived HSC self-renewal capacity upon introduction of oncogenes in order to provide further insights into MPN stem cell generation. DisclosuresBarroga: Wintherix: Employment. Jamieson: Merck: Research Funding; Pfizer: Research Funding; Wintherix: Consultancy; TargeGen: Research Funding; Celgene: Research Funding; Coronado Biosciences: Research Funding.

Regulation of Reactive Oxygen Species and Genomic Stability in Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are defined by their ability both to self-renew and to give rise to fresh blood cells throughout the lifetime of an animal. The failure of HSCs to self-renew during aging is believed to depend on several intrinsic (cell-autonomous) and extrinsic (non-cell-autonomous) factors. In this review, we focus on how dysregulation of reactive oxygen species (ROS) and disruptions of genomic stability can impair HSC functions. Recently, it was shown that long-term self-renewing HSCs normally possess low levels of intracellular ROS. However, when intracellular ROS levels become excessive, they cause senescence or apoptosis, resulting in a failure of HSC self-renewal. Repression of intracellular ROS levels in HSCs by treatment with an antioxidant that scavenges ROS can rescue HSC functions, indicating that excess ROS levels are at the root of HSC failure. Products of numerous genes that are involved in either DNA-damage responses or longevity-related signaling contribute to the maintenance of the HSC self-renewal capacity. Further investigations on the molecular mechanisms of ROS regulation and on the manipulation of excess ROS levels could lead to the development of novel therapeutics for hematopoietic diseases, regenerative medicine, and the prevention of leukemia.

Cyclin C Regulates the Quiescence of Human CD34+CD38- Hematopoietic Stem Cells.

The relative quiescence of adult hematopoietic stem cells (HSCs) at steady state represents an important regulatory mechanism for maintaining their self-renewal and engraftment capacity, as well as their resistance to cytotoxic insults. However, the specific mechanisms regulating the intermittent entry of HSCs into the cell cycle are not well characterized. Here we provide the evidence that cyclin C (CCNC) specifically promotes the G0/G1 transition of human CD34+CD38- HSCs, and thus can significantly affect the loss of HSC self-renewal capacity in in vitro culture. Based on the recently hypothesized specific function of CCNC in G0 exit of human fibroblasts, we have analyzed the effects of CCNC loss on the behavior of human cord blood HSCs. We achieved a highly efficient knockdown of CCNC expression (>90%) using lentiviral shRNA (shCCNC) transduction of freshly isolated human cord blood CD34+ cells, allowing the in vitro assessment of early cell cycle regulation in HSCs. First, we observed a 3-fold increase in the G0 fraction of shCCNC transduced CD34+ cells compared to the empty vector control, based on the Pyronin Y and Hoechst 33342 staining 72h after infection. The depletion of CCNC did not prevent cell cycle progression beyond the G1 entry, as we observed no significant changes in the G1/S/G2-M distribution, indicating that critical CCNC activities may be restricted to the G0 checkpoint. Consistent with the reported enrichment of functional HSCs in the G0 fraction, CCNC knockdown (CCNC KD) cells showed increased activity in all surrogate in vitro assays of stem cell-ness tested: a ∼3 fold increase in CD34+ population after long term culture, a ∼2.5 fold increase in long-term culture initiating cells (LTC-ICs) and a ∼3.5 fold increase in cobblestone area forming cells (CAFCs). In contrast, CFU assays using freshly sorted shCCNC cells (and cells obtained after one-week culture in cytokines) showed only a minimal decrease in total colony number, with no difference in colony composition or morphology, indicating no significant effect on hematopoietic progenitor cell differentiation. However, we did observe a prominent effect on secondary CFUs after 2 and 3 weeks in liquid culture (i.e. using the delta assay), namely a 2-fold and 30-fold increase in shCCNC over control culture respectively, again indicating a specific function of CCNC on the more primitive cells. Consistently, CCNC KD robustly enhanced CD34 expression and secondary CFU maintenance in sorted CD34+CD38- cells (HSCs); both markers of hematopoietic cell immaturity were rapidly lost in CD34+CD38+ cells (i.e. the committed progenitor cells) with no detectable effect of shCCNC transduction. Finally, we have found that these effects of CCNC depletion are likely the result of its initial loss of function, as transient CCNC KD, using siRNA transfection of CD34+ cells, produced similar biological effects as the constitutive lentiviral shCCNC expression. Collectively, these data indicate a cell context-dependent effect of CCNC KD on the initial rate of cell cycle entry by quiescent HSCs and suggest that this approach could be used to preserve their functional capacity in culture, potentially enhancing the ex vivo expansion of HSCs, as well as their use in gene therapy protocols. Transplantation of transduced CD34+ cells into sublethally irradiated immunodeficient mice is now under way to establish the potentially beneficial effects of CCNC KD on the engraftment and repopulating capability of cultured HSCs.

Smad7 promotes self-renewal of hematopoietic stem cells

AbstractThe Smad-signaling pathway downstream of the transforming growth factor–β superfamily of ligands is an evolutionarily conserved signaling circuitry with critical functions in a wide variety of biologic processes. To investigate the role of this pathway in the regulation of hematopoietic stem cells (HSCs), we have blocked Smad signaling by retroviral gene transfer of the inhibitory Smad7 to murine HSCs. We report here that the self-renewal capacity of HSCs is promoted in vivo upon blocking of the entire Smad pathway, as shown by both primary and secondary bone marrow (BM) transplantations. Importantly, HSCs overexpressing Smad7 have an unperturbed differentiation capacity as evidenced by normal contribution to both lymphoid and myeloid cell lineages, suggesting that the Smad pathway regulates self-renewal independently of differentiation. Moreover, phosphorylation of Smads was inhibited in response to ligand stimulation in BM cells, thus verifying impairment of the Smad-signaling cascade in Smad7-overexpressing cells. Taken together, these data reveal an important and previously unappreciated role for the Smad-signaling pathway in the regulation of self-renewal of HSCs in vivo.

Foxo3a Is Essential for the Quiescence and Self-Renewal of Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state in bone marrow (BM). Quiescent stem cells show resistance to various stresses, suggesting that mechanisms for protection of HSC life from stress contribute to maintenance of self-renewal capacity through a whole life in animals. We hypothesized that a signaling pathway for regulating aging might be involved in stem cell functions.FOXO transcription factors belong to the forkhead family of transcriptional regulators characterized by a conserved DNA-binding domain termed “forkhead box”. In C.elegans, genetic analyses have revealed the existence of a conserved insulin-like signaling involved in longevity. Conservation of this pathways lead to speculation that forkhead transcriptional factor are involved in life span in mammals. It was known that active-state Foxo3a is localized in nucleus, and we found HSC-specific nuclear localization of Foxo3a by immunocytochemistric study, therefore we generated gene-targeted Foxo3a−/− mice to analyze roles of Foxo in HSC regulation. Peripheral blood count showed decreased number of red blood cells in Foxo3a−/− mice, but numbers of white blood cells and platelets were normal. In colony-forming assay, we detected the numbers and sizes of myeloid, erythroid and mixed colonies derived from Foxo3a−/− BM mononuclear cells were all normal. These results suggest that the proliferation and differentiation of Foxo3a−/− progenitors were normal. However, the number of colony-forming cells present in long-term culture of Foxo3a−/− c-kit+Sca-1+Lin− (KSL) cells with stroma was significantly reduced. The ability of Foxo3a−/− HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired, indicating that self-renewal capacity of HSCs was defective in Foxo3a−/− mice. To understand the mechanisms of this phenotypes, we evaluated the cell cycle status using BrdU (5-bromodeoxyuridine) incorporation but found no difference in Foxo3a+/+ and Foxo3a−/− progenitor cells. To directly evaluate HSC quiescence in Foxo3a−/− mice, we stained CD34−KSL cells with Pyronin Y. Although most Foxo3a+/+ CD34−KSL cells stained negatively for Pyronin Y, a sizable Pyronin Y+ population was detected among Foxo3a−/− CD34−KSL cells, demonstrating that loss of Foxo3a leads to a defect in the maintenance of HSCs quiescence. Since p38MAPK is selectively activated by environmental stress, we evaluated the activation status of p38MAPK in Foxo3a+/+ and Foxo3a−/− HSCs. Frequency of phosphorylated p38MAPK+ cells in Foxo3a−/−CD34−KSL cells was significantly increased than that of Foxo3a+/+CD34−KSL cells. Our results suggest that Foxo3a−/− HSCs subjected to tangible stress in vivo. Finally, we investigated the sensitivity of Foxo3a−/− mice to weekly 5-fluorouracil treatment in vivo. Although 60% of Foxo3a+/+mice survived for at least 4 weeks post-injection, all Foxo3a−/− mice were dead in 4 weeks. It suggests that Foxo3a protects hematopoietic cells from destruction by cell cycle-dependent myelotoxic agent. Taken together, our results demonstrate that Foxo3a plays a pivotal role in maintaining HSC quiescence and stress resistance.

Isolation and clonal characterization of hematopoietic and liver stem cells.

Prospective isolation of stem cells is essential to understanding the mechanisms that control their proliferation and differentiation. Using 9 monoclonal antibodies and fluorescence-activated cell sorting (FACS), we have succeeded in prospectively identifying hematopoietic stem cells (HSCs) in adult mouse bone marrow. Mouse HSCs were exclusively enriched in CD34 negative, c-Kit Sca-1 Lineage Marker (CD34 KSL) cells representing 0.004% of bone marrow (BM) mononuclear cells. When single CD34-KSL cells were transplanted individually into a lethally irradiated mouse, 25% of the recipient mice survived and showed long-term reconstitution of the BM, providing evidence for multipotency and a self-renewal capacity of HSCs. Using a similar approach, we also prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewal capability in the c-Met CD49f c-Kit CD45 Ter119 fraction of cells isolated from day 13.5 fetal mouse liver. On cell transplantation, these cells differentiated into hepatocytes and cholangiocytes. As an alternative to the antibody based stem cell isolation, Hoechst33342 staining is useful. To understand the mechanism responsible for SP phenotype, we performed an expression cloning and identified bcrp-1/ABCG2 gene, a member of ATP binding-cassette (ABC) transporter family. Bcrp-1 is almost exclusively expressed in CD34 KSL cells among blood cells; however their expression in other tissue specific stem cells remains to be studied. With the use of FACS and monoclonal antibodies, hematopoietic and liver stem cells were prospectively isolated and characterized. HSCs could also be purified by Hoechst 33342 staining. By expression cloning, we identify bcrp-1/ABCG2 transporter as a molecule responsible for SP phenotype. Elucidation of the physiological role of bcrp-1/ABCG2 in HSCs may provide us with clues to understand the molecular mechanisms of stem cell self-renewal and differentiation.

The elements of stem cell self-renewal: a genetic perspective.

Every day, the body produces billions of new blood cells. Each of these is derived from a rare cell in the bone marrow called the hematopoietic stem cell (HSC). Because most mature blood cells have a limited lifespan, the ability of HSCs to self-renew and replenish the mature cell compartment is critical to sustaining life. While great progress has been made in isolating HSCs and defining their functional and phenotypic characteristics, the molecular mechanisms that regulate their self-renewal remain a mystery. Over the last few years, alterations in HSC frequency and self-renewal capacity in transgenic and knock-out mice have led to the identification of novel mediators of HSC homeostasis in vivo. These genetically modified mice have revealed that maintenance of survival, proliferation, quiescence, and normal telomere length all contribute to the self-renewal of HSCs. They also highlight the need to test in context of the normal microenvironment the role of signaling molecules such as Notch and Wnt, which have emerged recently as important regulators of HSC self-renewal. The emerging picture these data provide of the regulation of self-renewal in HSCs has provided a better understanding of the basic biology of stem cells and holds promise for designing strategies to improve bone marrow transplantation.

The variation of hematopoietic stem cell self-renewal capacity as a function of age: further evidence for heterogenicity of the stem cell compartment

The variation of hematopoietic stem cell self-renewal capacity as a function of age: further evidence for heterogenicity of the stem cell compartment

The Variation of Hematopoietic Stem Cell Self-Renewal Capacity as a Function of Age: Further Evidence for Heterogenicity of the Stem Cell Compartment

There is substantial evidence for limited proliferative capacity of the hematopoietic progenitors. Hematopoiesis in mice is maintained by primitive stem cells that are assayed by their ability to form spleen colonies (CFU-S). To measure the average proliferative capacity of these cells (CFU-S), bone marrow containing 50-150 CFU-S is injected into lethally irradiated mice, and 14 days later, the number present in the recipient marrow is measured. In fetal life between 13 and 16 days of gestation, the hematopoietic stem cells are primarily localized in the liver. There is a decrease in the average proliferative capacity of these stem cells as they progress from fetal life into adulthood. This proliferative capacity of these significantly increases in late adulthood. However, if one scores maximum self-renewal or proliferative capacity by another assay consisting of the serial transplantation of marrow at 14-day intervals, this capacity is unchanged with age. These observations can be explained by considering the stem cell compartment as a continuum of cells whose self-renewal capacity varies inversely with their divisional history. All stem cells participate in repopulation when the pool needs rapid expansion (fetal life). During steady-state repopulation (adult life), clones are selected on the basis of their divisional history, those with the greatest proliferative history being selected. Thus, the average proliferative capacity of the remaining stem cells will increase as the adult animal ages, since stem cells of low proliferative potential have been consumed. The maximal self-renewal capacity will remain unchanged, since this measures the spared earliest cells with the highest proliferative capacity.