Charlie Mantel

SIRT1 regulates apoptosis and Nanog expression in mouse embryonic stem cells by controlling p53 subcellular localization

Nuclear tumor suppressor p53 transactivates proapoptotic genes or antioxidant genes depending on stress severity, while cytoplasmic p53 induces mitochondrial-dependent apoptosis without gene transactivation. Although SIRT1, a p53 deacetylase, inhibits p53-mediated transactivation, how SIRT1 regulates these p53 multifunctions is unclear. Here we show that SIRT1 blocks nuclear translocation of cytoplasmic p53 in response to endogenous reactive oxygen species (ROS) and triggers mitochondrial-dependent apoptosis in mouse embryonic stem (mES) cells. ROS generated by antioxidant-free culture caused p53 translocation into mitochondria in wild-type mES cells but induced p53 translocation into the nucleus in SIRT1(-/-) mES cells. Endogenous ROS triggered apoptosis of wild-type mES through mitochondrial translocation of p53 and BAX but inhibited Nanog expression of SIRT1(-/-) mES, indicating that SIRT1 makes mES cells sensitive to ROS and inhibits p53-mediated suppression of Nanog expression. Our results suggest that endogenous ROS control is important for mES cell maintenance in culture.

Stromal cell-derived factor-1/CXCL12 directly enhances survival/antiapoptosis of myeloid progenitor cells through CXCR4 and Gαi proteins and enhances engraftment of competitive, repopulating stem cells

Stromal cell‐derived factor‐1 (SDF‐1/CXCL12) enhances survival of myeloid progenitor cells. The two main questions addressed by us were whether these effects on the progenitors were direct‐acting and if SDF‐1/CXCL12 enhanced engrafting capability of competitive, repopulating mouse stem cells subjected to short‐term ex vivo culture with other growth factors. SDF‐1/CXCL12 had survival‐enhancing/antiapoptosis effects on human bone marrow (BM) and cord blood (CB) and mouse BM colony‐forming units (CFU)‐granulocyte macrophage, burst‐forming units‐erythroid, and CFU‐granulocyte‐erythroid‐macrophage‐megakaryocyte with similar dose responses. The survival effects were direct‐acting, as assessed on colony formation by single isolated human BM and CB CD34+++ cells. Effects were mediated through CXCR4 and Gαi proteins. Moreover, SDF‐1/CXCL12 greatly enhanced the engrafting capability of mouse long‐term, marrow‐competitive, repopulating stem cells cultured ex vivo with interleukin‐6 and steel factor for 48 h. These results extend information on the survival effects mediated through the SDF‐1/CXCL12–CXCR4 axis and may be of relevance for ex vivo expansion and gene‐transduction procedures.

Transgenic expression of stromal cell-derived factor-1/CXC chemokine ligand 12 enhances myeloid progenitor cell survival/antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo.

Hemopoiesis is regulated in part by survival/apoptosis of hemopoietic stem/progenitor cells. Exogenously added stromal cell-derived factor-1 ((SDF-1)/CXC chemokine ligand (CXCL)12) enhances survival/antiapoptosis of myeloid progenitor cells in vitro. To further evaluate SDF-1/CXCL12 effects on progenitor cell survival, transgenic mice endogenously expressing SDF-1/CXCL12 under a Rous sarcoma virus promoter were produced. Myeloid progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte) from transgenic mice were studied for in vitro survival in the context of delayed addition of growth factors. SDF-1-expressing transgenic myeloid progenitors were enhanced in survival and antiapoptosis compared with their wild-type littermate counterparts. Survival-enhancing effects were due to release of low levels of SDF-1/CXCL12 and mediated through CXCR4 and Gαi proteins as determined by ELISA, an antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin. Transgenic effects of low SDF-1/CXCR4 may be due to synergy of SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes with low concentrations of other cytokines to enhance survival of normal mouse myeloid progenitors. Consistent with in vitro results, progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow and splenic myelopoiesis: greatly increased progenitor cell cycling and significant increases in progenitor cell numbers. These results substantiate survival effects of SDF-1/CXCL12, now extended to progenitors engineered to endogenously produce low levels of this cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition to cell trafficking.

Dipeptidylpeptidase 4 negatively regulates colony-stimulating factor activity and stress hematopoiesis

Enhancement of hematopoietic recovery after radiation, chemotherapy, or hematopoietic stem cell (HSC) transplantation is clinically relevant. Dipeptidylpeptidase (DPP4) cleaves a wide variety of substrates, including the chemokine stromal cell-derived factor-1 (SDF-1). In the course of experiments showing that inhibition of DPP4 enhances SDF-1–mediated progenitor cell survival, ex vivo cytokine expansion and replating frequency, we unexpectedly found that DPP4 has a more general role in regulating colony-stimulating factor (CSF) activity. DPP4 cleaved within the N-termini of the CSFs granulocyte-macrophage (GM)-CSF, G-CSF, interleukin-3 (IL-3) and erythropoietin and decreased their activity. Dpp4 knockout or DPP4 inhibition enhanced CSF activities both in vitro and in vivo. The reduced activity of DPP4-truncated versus full-length human GM-CSF was mechanistically linked to effects on receptor-binding affinity, induction of GM-CSF receptor oligomerization and signaling capacity. Hematopoiesis in mice after radiation or chemotherapy was enhanced in Dpp4−/− mice or mice receiving an orally active DPP4 inhibitor. DPP4 inhibition enhanced engraftment in mice without compromising HSC function, suggesting the potential clinical utility of this approach.

Flt3 signaling involves tyrosyl-phosphorylation of SHP-2 and SHIP and their association with Grb2 and Shc in Baf3/Flt3 cells

Flt3 ligand (FL) is an early‐acting potent co‐stimulatory cytokine that regulates proliferation and differentiation of a number of blood cell lineages. Its receptor Flt3/Flk2 belongs to class III receptor tyrosine kinases that also include the receptors for colony‐stimulating factor 1, Steel factor, and platelet‐derived growth factor. Using CSF‐1 receptor/Flt3 chimeras, two groups have characterized some of the post‐receptor signaling events and substrate specificity of murine Flt3 receptor. However, there are few studies on the signaling pathway through human Flt3. We examined human Flt3 signaling pathways in a murine IL‐3‐dependent hematopoietic cell line Baf3, which stably expresses full‐length human Flt3 receptor. This subline proliferates in response to human FL. Like the chimeric murine Flt3, human Flt3 undergoes autophosphorylation, associates with Grb2, and leads to tyrosine phosphorylation of Shc on ligand binding. We found that SHP‐2, but not SHP‐1, is tyrosine‐phosphorylated by FL stimulation. SHP‐2 does not associate with Flt3, but binds directly to Grb2. SHIP is also tyrosine‐phosphorylated and associates with Shc after FL simulation. We further examined the downstream signaling pathway. FL transiently activates MAP kinase. This activation could be blocked by PD98059, a specific MEK inhibitor. PD98059 also blocked cell proliferation in response to FL. These results demonstrate that SHP‐2 and SHIP are important components in the human Flt3 signaling pathway and suggest that SHP‐2 and SHIP, by forming complexes with adapter proteins Grb2 and Shc, may modulate MAP kinase activation, which may be necessary for the mitogenic signaling of Flt3. J. Leukoc. Biol. 65: 372–380; 1999.

Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

Oct‐4 Is Critical for Survival/Antiapoptosis of Murine Embryonic Stem Cells Subjected to Stress: Effects Associated with Stat3/Survivin

Understanding survival/antiapoptosis of murine embryonic stem (ES) cells may enhance their clinical potential. We hypothesized that Oct‐4 might be involved in survival of undifferentiated ES cells under stress. The Oct‐4 tetracycline conditional knockout cell line ZHBtc4 was used to test this possibility, and apoptosis was induced by either etoposide, heat shock, or UV exposure. Apoptosis in Oct‐4 knocked‐down ES cells was significantly increased in response to all stress situations compared with parental cells. Oct‐4 knockdown was not associated with changes in morphology or expression of Nanog, SSEA‐1, KLF‐4, or Sox2 within the time frame and culture conditions used, suggesting that enhanced sensitivity of these cells to apoptosis was not due to an overtly differentiated state of the cells. To address potential intracellular mediators, we focused on the inhibitor of apoptosis proteins family member Survivin, an antiapoptosis protein. The Survivin promoter was transfected into ES cells after knockdown of Oct‐4. Survivin promoter activity was dramatically decreased in the Oct‐4 knockdown cells. Western blots substantiated that Oct‐4 knockdown ES cells had decreased Survivin protein expression. Since the Survivin promoter does not have binding sites for Oct‐4, this suggested an indirect effect of Oct‐4 on expression of Survivin. Leukemia inhibitory factor‐induced signal transducer and activator of transcription‐3 (STAT3) is responsible for ES cell survival, and STAT3 regulates Survivin expression in breast cancer cells. Western blot analysis showed that downregulated Oct‐4 was associated with decreased phosphorylation of STAT3. Our results suggest that Oct‐4 is essential for antiapoptosis of ES cells in response to stress, effects that may be mediated through the STAT3/Survivin pathway.

AMD3100 and CD26 Modulate Mobilization, Engraftment, and Survival of Hematopoietic Stem and Progenitor Cells Mediated by the SDF‐1/CXCL12‐CXCR4 Axis

Abstract:  The chemokine stromal cell‐derived factor‐1 (SDF‐1/CXCL12) and its receptor, CXCR4, are involved in a number of facets of the regulation of hematopoiesis at the level of hematopoietic stem (HSCs) and progenitor (HPCs) cells. Modulation of this ligand–receptor interaction may be of clinical utility. We now report that: (1) the CC chemokine, macrophage inflammatory protein‐1α (MIP‐1α/CCL3) synergizes with AMD3100 (an antagonist of the binding of SDF‐1/CXCL12 to CXCR4) to rapidly mobilize HPCs to the blood of mice; moreover, the combination of granulocyte colony‐stimulating factor (G‐CSF) with AMD3100 and MIP‐1α/CCL3, given in a specific sequence, mobilizes the greatest number of HPCs compared to any combination of two of these mobilizing agents; (2) pretreatment of recipient mice with Diprotin A, an inhibitor of CD26/Dipeptidylpeptidase IV (DPPIV), enhances the competitive HSCs repopulating capacity of untreated donor cells; (3) the survival‐enhancing effects of SDF‐1/CXCL12 on HPCs subjected in vitro to delayed addition of growth factors (GFs) are mediated in part through the cell cycle‐related proteins p21cip1/waf1 (as assessed using p21cip1/waf1−/− and +/+ mice) and Mad2 (using Mad2 +/− and +/+ mice); and (4) deletion of CD26/DPPIV on mouse bone marrow cells increases the survival‐enhancing effects of SDF‐1/CXCL12 on HPCs. These results demonstrate the means to increase the mobilization of HPCs, the engrafting capability of HSCs, and responsiveness of HPCs to the survival‐enhancing activity of SDF‐1/CXCL12, effects that may be of practical value.

Human chemokines: enhancement of specific activity and effects in vitro on normal and leukemic progenitors and a factor-dependent cell line and in vivo in mice.

The myelosuppressive effects of human chemokines were evaluated in vitro on normal myeloid progenitors obtained from bone marrow and cord blood, on bone marrow progenitors from patients with acute or chronic leukemia, on proliferation of human factor-dependent cell line M07e, and in vivo on myelopoiesis in mice. Preincubation of human MIP-1α, MIP-2α, interleukin (IL)-8, platelet factor (PF) 4, monocyte chemotactic and activating factor (MCAF), and interferon-inducible protein-10 (IP-10) in an acetonitrile (ACN) solution significantly enhanced the specific activity of these chemokines for in vitro suppression of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells stimulated to proliferate with a colony stimulating factor plus steel factor (SLF). Combinations of any two of these ACN-treated chemokines synergized to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM at chemokine concentrations below that at which combinations of non-ACN treated chemokines are active. Cord blood progenitors, as previously reported, were in a slow or noncycling state and nonresponsive to inhibition by chemokines. However, after suspension culture with GM-CSF, IL-3, and SLF, they were placed into rapid cell cycle and were responsive to inhibition by ACN-treated chemokines. Low doses of these ACN-pretreated chemokines were active in vivo in suppressing absolute numbers and cycling status of femoral marrow CFU-GM, BFU-E, and CFU-GEMM in C3H/HeJ mice. Other chemokines, alone and in combination, including MIP-1β, MIP-2β, GRO-α NAP-2, and RANTES, were inactive in vitro and in vivo whether or not they were pretreated with ACN. While heterogeneity in responsiveness of CFU-GM from different patients with leukemia to suppression by ACN-treated chemokines was apparent, if the patients had CFU-GM responsive to one of the active chemokines these cells were responsive to the other active chemokines; if patient CFU-GM were not responsive to one of the chemokines, they were not responsive to the other active chemokines. M07e colony-forming cells were responsive to the growth-inhibiting effects of the active ACN-treated chemokines, alone and in combination, but these effects were rapidly reversible and sustained only by multiple daily additions of chemokines. These results should be of value in considering these chemokines for potential clinical use and for assessment of their mechanisms of action, alone and in combination.

Epigenetic Regulation of Nanog by MiR-302 Cluster-MBD2 Completes Induced Pluripotent Stem Cell Reprogramming

While most somatic cells undergoing induced pluripotent stem (iPS) cell reprogramming with Yamanaka factors accumulate at stable partially reprogrammed stages, the molecular mechanisms required to achieve full reprogramming are unknown. MicroRNAs (miRNAs) fine‐tune mRNA translation and are implicated in reprogramming, but miRNA functional targets critical for complete iPS cell reprogramming remain elusive. We identified methyl‐DNA binding domain protein 2 (MBD2) as an epigenetic suppressor, blocking full reprogramming of somatic to iPS cells through direct binding to NANOG promoter elements preventing transcriptional activation. When we overexpressed miR‐302 cluster we observed a significant increase in conversion of partial to fully reprogrammed iPS cells by suppressing MBD2 expression, thereby increasing NANOG expression. Thus, expression of exogenous miR‐302 cluster (without miR‐367) is efficient in attaining a fully reprogrammed iPS state in partially reprogrammed cells by relieving MBD2‐mediated inhibition of NANOG expression. Our studies provide a direct molecular mechanism involved in generating complete human iPS cell reprogramming to study disease pathogenesis, drug screening, and for potential cell‐based therapies. STEM CELLS 2013;31:666–681