William Blanco-Bose

Hematopoietic Stem Cells Reversibly Switch from Dormancy to Self-Renewal during Homeostasis and Repair

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

IFNα activates dormant haematopoietic stem cells in vivo.

Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs) with long-term self-renewal capacity. After injury these cells are induced to proliferate to quickly re-establish homeostasis. The signalling molecules promoting the exit of HSCs out of the dormant stage remain largely unknown. Here we show that in response to treatment of mice with interferon-α (IFNα), HSCs efficiently exit G0 and enter an active cell cycle. HSCs respond to IFNα treatment by the increased phosphorylation of STAT1 and PKB/Akt (also known as AKT1), the expression of IFNα target genes, and the upregulation of stem cell antigen-1 (Sca-1, also known as LY6A). HSCs lacking the IFNα/β receptor (IFNAR), STAT1 (ref. 3) or Sca-1 (ref. 4) are insensitive to IFNα stimulation, demonstrating that STAT1 and Sca-1 mediate IFNα-induced HSC proliferation. Although dormant HSCs are resistant to the anti-proliferative chemotherapeutic agent 5-fluoro-uracil, HSCs pre-treated (primed) with IFNα and thus induced to proliferate are efficiently eliminated by 5-fluoro-uracil exposure in vivo. Conversely, HSCs chronically activated by IFNα are functionally compromised and are rapidly out-competed by non-activatable Ifnar-/- cells in competitive repopulation assays. Whereas chronic activation of the IFNα pathway in HSCs impairs their function, acute IFNα treatment promotes the proliferation of dormant HSCs in vivo. These data may help to clarify the so far unexplained clinical effects of IFNα on leukaemic cells, and raise the possibility for new applications of type I interferons to target cancer stem cells.

Lentiviral-mediated RNA interference

RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA). These siRNAs are capable of binding to a specific mRNA sequence and causing its degradation. The recent demonstration of a plasmid vector that directs siRNA synthesis in mammalian cells prompted us to examine the ability of lentiviral vectors to encode siRNA as a means of providing long-term gene silencing in mammalian cells. The RNA-polymerase III dependent promoter (H1-RNA promoter) was inserted in the lentiviral genome to drive the expression of a small hairpin RNA (shRNA) against enhanced green fluorescent protein (EGFP). This construct successfully silenced EGFP expression in two stable cell lines expressing this protein, as analyzed by fluorescence microscopy, flow cytometry, and Western blotting. The silencing, which is dose dependent, occurs as early as 72 hr postinfection and persists for at least 25 days postinfection. The ability of lentiviruses encoding siRNA to silence genes specifically makes it possible to take full advantage of the possibilities offered by the lentiviral vector and provides a powerful tool for gene therapy and gene function studies.

Hematopoietic Stem Cell Function and Survival Depend on c-Myc and N-Myc Activity

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule Granzyme B, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

Dormant and Self‐Renewing Hematopoietic Stem Cells and Their Niches

Abstract:  In the mouse, over the last 20 years, a set of cell‐surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long‐term self‐renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single‐cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c‐Kit, CD135, CD48, CD150, and CD34) on lineage‐depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin−Sca1+c‐Kit+ CD48−CD150+CD34−) that gives rise to an active self‐renewing CD34+ HSC population. HSC dormancy, as well as the balance between self‐renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

c-Myc and its target foxM1 are critical downstream effectors of constitutive androstane receptor (CAR) mediated direct liver hyperplasia

In the adult liver, 1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene (TCPOBOP), an agonist of the constitutive androstane receptor (CAR, NR1I3), produces rapid hepatomegaly in the absence of injury. In this study, we identify c‐Myc as a gene induced by CAR and demonstrate that TCPOBOP‐induced proliferation of hepatocytes depends on c‐Myc function. Moreover, the TCPOBOP‐induced cell cycle program (Cdc2, cyclins, MCM proteins, Cdc20, and genes implicated in the spindle assembly checkpoint) is severely impaired in c‐Myc mutant livers. Strikingly, many of these genes overlap with a program controlled by the forkhead transcription factor FoxM1, known to control progression through S‐phase and mitosis. Indeed, FoxM1 is also induced by TCPOBOP. Moreover, we show that c‐Myc binds to the FoxM1 promoter in a TCPOBOP‐dependent manner, suggesting a CAR → c‐Myc → FoxM1 pathway downstream of TCPOBOP. Conclusion: Collectively, this study identifies c‐Myc and FoxM1 mediated proliferative programs as key mediators of TCPOBOP‐CAR induced direct liver hyperplasia. (HEPATOLOGY 2008.)

Pten loss in the bone marrow leads to G-CSF–mediated HSC mobilization

Loss of the phosphatase and tumor suppressor gene PTEN induces G-CSF production in myeloid and stromal cells, thereby promoting HSCs mobilization from the bone marrow to the spleen and the initiation of lethal leukemia.