scholarly journals Altered HSC Metabolism in Response to Stress Leads to De Novo dna Damage and Cellular Attrition

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 255-255
Author(s):  
Anja Geiselhart ◽  
Dagmar Walter ◽  
Amelie Lier ◽  
Frederic B. Thalheimer ◽  
Sina Huntscha ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Cell Death ◽  
De Novo ◽  
Bone Marrow Failure ◽  
Fold Increase ◽  
Quiescent State ◽  
Response To Stress

Abstract Hematopoietic stem cells (HSCs) reside in a quiescent state, which is thought to preserve their genomic stability during aging. HSCs are forced to exit this so-called dormant state and enter into cycle in response to stress stimuli such as infections or severe bleeding. This situation may provoke high levels of proliferative stress in HSCs and a subsequent decline in stem cell function. We recently found that de novo DNA damage can be precipitated in HSCs in vivo by enforcing cell cycle progression using agonists that mimic physiologic stress, such as interferons, G-CSF, TPO or serial bleeding, (Walter et al., Blood, 122, 21:799). The Fanconi anemia (FA) DNA repair pathway is an important route via which this replication damage is resolved in HSCs in vivo. In FA deficient mice, DNA damage repair was impaired, provoking HSC depletion and severe aplastic anemia (p<0.01) upon serial treatment with the synthetic double-stranded RNA mimetic polyI:polyC (pI:pC). Here, we sought to identify the mechanistic basis of the stress-induced DNA damage acquisition and concomitant HSC attrition in vivo. Activated HSCs exhibited elevated mitochondrial membrane potential, indicative of increased energy production via oxidative phosphorylation (>2-fold increase, p<0.01). Next, to determine whether there was an associated increase in intracellular reactive oxygen species (ROS) production, we made use of genetically encoded fluorescent biosensors to detect the status of specific redox couples within different HSC compartments in vivo. Activated HSCs demonstrated increased levels of oxidized mitochondrial glutathione (2.3-fold increase, p<0.01) and cytoplasmic hydrogen peroxide (1.6-fold increase, p<0.05) compared to dormant HSC controls. These enhanced ROS levels directly correlated with elevated 8-Oxo-dG lesions on the DNA of HSCs that had been activated into cycle in vivo(>1.3-fold increase, p<0.05). Finally, retroviral over-expression of ROS-detoxifying enzymes completely rescued gH2AX foci formation in cycling HSCs, demonstrating a direct functional link between stress-induced DNA damage and altered redox biology. We next performed live cell video imaging on individual WT and Fanca-/- LT-HSCs in vitro in order to track cell fate decisions upon exit from quiescence. In the first division upon exit from quiescence, Fanca-/- HSCs were frequently observed to undergo abnormal mitoses while this was not evident in WT HSCs. At this time point, we observed elevated DNA damage in Fanca-/- HSCs as measured by gH2AX, 53BP1 and RAD51 foci, as well as increased ROS-induced 8-Oxo-dG lesions (>5-fold increase, p<0.01). HSCs from Fanca-/- mice demonstrated a significantly higher rate of replication-dependent cell death following the first division (24% vs. 6%, p<0.05%) suggesting that apoptosis is the major route via which HSCs are lost in response to stress-induced DNA damage. Taken together, these data strongly implicate stress-induced exit from dormancy as a cause of physiologic DNA damage in HSCs in vivo. Under stress conditions, the increased energy demand of cycling stem cells leads to elevated levels of ROS in mitochondria and cytoplasm, which is a direct source of DNA damage. If unresolved by the FA-dependent DNA damage response, this DNA damage accumulates in the cell and provokes apoptotic cell death. This recapitulates the highly penetrant bone marrow failure syndrome in FA patients and suggests that their HSCs are lost due to an aberrant response to HSC activation, most likely as a consequence of infection or other physiologic stress. These data provide a novel link between stress hematopoiesis, ROS, DNA damage and HSC loss and may have important clinical implications in the study of age-related hematopoietic defects in both FA and non-FA patients. Moreover, these data provide the first evidence that FA knockout mouse models can be utilized to accurately recapitulate the etiology of bone marrow failure through the progressive application of stress-induced alterations in HSC function that mimic usual physiologic stressors such as infection. Disclosures No relevant conflicts of interest to declare.

HSC Exit From Dormancy Provokes De Novo DNA Damage, Leading To Bone Marrow Failure If Unresolved By The Fanconi Anemia Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 799-799
Author(s):  
Dagmar Walter ◽  
Amelie Lier ◽  
Anja Geiselhart ◽  
Sina Huntscha ◽  
David Brocks ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Aplastic Anemia ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
De Novo ◽  
Bone Marrow Failure ◽  
Damage Response ◽  
Fold Induction

Abstract Long-term quiescence has been proposed to preserve the genomic stability of hematopoietic stem cells (HSCs) during aging. The current models of HSC aging are limited in their ability to observe both DNA damage in vivo and the consequences of this damage upon hematopoiesis. Fanconi Anemia (FA) is a hereditary multisystem disorder, characterized by defective DNA damage response and progressive bone marrow failure in most patients. However, the existing genetic models of FA do not develop aplastic anemia, suggesting that cell-extrinsic factors may play a causal role. We sought to identify whether physiologic mediators of HSC activation could be used as agonists to provoke DNA damage and HSC attrition in vivo. Mice were treated with a range of agonists that promote the in vivo exit of HSC from a dormant state into active cycling (polyI:polyC; Interferon-α; G-CSF; TPO; and serial bleeding). Highly purified HSC demonstrated a rapid 3-5-fold induction of DNA damage after treatment with all agonists (p<0.01), as assessed by both enumerating γ-H2AX foci and by alkaline comet assay. Mechanistically, stress-induced exit from quiescence correlated with increased mitochondrial metabolism in HSC, as evaluated by elevated mitochondrial membrane potential (2-fold increased, p<0.01) and superoxide levels (1.5-fold increased, p<0.05). Critically, we could directly implicate these reactive oxygen species in DNA damage as we observed a 1.4-fold increase in 8-Oxo-dG lesions in HSC that had been activated into cycle in vivo(p<0.05). At 48 h post-treatment, γ-H2AX levels began to decrease and this repair was concomitant with an induction of the FA signaling pathway in HSC, as demonstrated by both increased levels of FA gene expression and elevated FANCD2 foci (4-fold induction, p<0.01). Treatment of Fanca-/- mice with polyI:polyC led to a HSC proliferative response comparable to wild type (WT) mice but resulted in a 2-fold higher level of activation-induced DNA damage (p<0.05), demonstrating that this repair pathway is involved in resolving activation-induced DNA damage. Four rounds of serial in vivo activation led to a permanent depletion of the most primitive label-retaining Fanca-/- HSC and this correlated with a 4-fold depletion of functional HSC (p<0.01) as defined by competitive repopulation assays. Subsequent rounds of HSC activation with polyI:polyC resulted in the onset of a severe aplastic anemia (SAA) in 33% of treated Fanca-/- mice but not in any of the WT controls. SSA was characterized by a dramatic reduction in bone marrow (BM) cellularity, profound thrombocytopenia (21-246x106 platelets/ml), leukocytopenia (0.4-0.5x106 WBC/ml), neutropenia (0.03-0.1x106/ml) and anemia (1.5-2.3 g/dL Hb). Examination of BM HSC/progenitors demonstrated nearly complete loss of HSC, MPP, CMP and CLP (depletion of ≥33x, 8x, 4x and 12x respectively compared to PBS-treated Fanca-/-controls). Taken together, these data demonstrates that enforced exit from dormancy in vivo leads to de novo DNA damage in HSC, which is repaired by activation of a FA-dependent DNA damage response. Furthermore, the highly penetrant bone marrow failure observed in Fanconi anemia patients can be recapitulated by the serial application of a physiologic HSC activating signal to Fanca-/- mice. This suggests that the BM failure in FA may be caused by an aberrant response to HSC activation, most likely during exposure to infection or other physiologic stressors. These data provides a novel link between pro-inflammatory cytokines, DNA damage and HSC dysfunction and may have important clinical implications relevant to both prevention of BM failure in FA and in the study of age-related hematopoietic defects in non-FA patients. Moreover, these data provide the first evidence that FA knockout mouse models accurately recapitulate and provide novel insights into the etiology of BM failure in patients with FA. Disclosures: No relevant conflicts of interest to declare.

Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1224-1224
Author(s):  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Actin Binding ◽  
Wild Type ◽  
Cell Fates ◽  
Hematopoietic Stem ◽  
Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Lentivirally Transduced Human Cord Blood CD34+FLT3-ITD+ Cells Induce Murine Acute Leukemia in the NOD/SCID Transplantation Model.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2984-2984
Author(s):  
Pawan Kaur Bollinger ◽  
Doris Steinemann ◽  
Rama Krishna Kancha ◽  
Leticia Quantanilla-Fend ◽  
Stefan K Bohlander ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Leukemia ◽  
Cord Blood ◽  
Fold Increase ◽  
Bone Marrow Niche ◽  
Human Cord Blood ◽  
Human Cd34 ◽  
Cord Blood Cells

Abstract Abstract 2984 Targeting constitutively activated FLT3 (FLT3-ITD) by tyrosine kinase inhibition (TKI) in acute myeloid leukemia (AML) leads to clearance of blasts in the periphery but not in the bone marrow, suggesting a protective effect of the marrow niche on leukemic stem cells (LSC). We have previously shown that interaction of CD34+FLT3-ITD+ LSC with stromal niche cells mimicking the bone marrow environment specifically protects these cells from the effects of TKI and confers a growth advantage to FLT3-ITD+ leukemic stem/progenitor cells over normal ones (Parmar et al, Cancer Research 2011). To study human FLT3-ITD+ LSC in vivo in the context of the bone marrow niche, we aimed to establish a xenogeneic NOD/SCID mouse model of human FLT3-ITD+AML. Human CD34+ enriched cord blood cells were transduced with a pWPI lentivirus containing a VSV-pseudotyped SIN/LTR vector with eGFP and full length human FLT3 cDNA harboring a 30 bp length internal tandem duplication (FLT3-ITD) or empty vector control. Transduction efficiency ranged between 1–4.4% for FLT3-ITD and 1.3–18% for vector control. Sub-lethally irradiated NOD/SCID mice were then transplanted with 1 × 104 – 7 × 104 unselected or GFP-sorted CD34+FLT3-ITD+ cells. Acute leukemia developed in 7/9 animals after a median latency of 85 days (range 70–168), with involvement of peripheral blood, bone marrow, spleen and liver. Three mice developed acute lymphoblastic leukemia (ALL) whereas the remaining mice showed signs of AML. In contrast, mice receiving empty pWPI vector-transduced human CD34+ cord blood cells (n = 8) all remained healthy during the observation period of 28 weeks and, in 4/8 animals, normal human CD34+cells could be recovered from the bone marrow (human engraftment range 0.3–35.5%). Leukemic mice exhibited hepatomegaly and splenomegaly with an average 10-fold increase in spleen weight, 2-fold increase in spleen length and 2.7-fold increase in liver weight compared to control mice. In mice that developed ALL, lymph node enlargement was also noted. Whole bone marrow, spleen and liver cells from primary mice were re-transplanted and were able to reproduce acute leukemia in all secondary (n=10/10) and tertiary mice (n=11/11) with a median latency of 25 and 20 days, respectively (p<0.01). Surprisingly, detailed immunophenotypical and immunohistochemical analysis revealed all leukemias to be of murine origin. Leukemic cells stained positively for murine CD45.1 antigen but negatively for human CD45. However, a small population of human CD34+CD45+ cells (range 1–7%) was continuously detectable in the bone marrow of primary, secondary and tertiary transplanted leukemic mice. Accordingly, human FLT3-ITD was detectable by PCR specific for human FLT3 up to the third serial transplantation. Viral integration site analysis by LM-PCR on genomic DNA isolated from spleens of leukemic mice revealed lentiviral integration into the human genome, excluding the possibility of in vivo viral shuttling from human cord blood CD34+ cells to mouse hematopoietic stem cells. Moreover, multicolor fluorescent in situ hybridization (M-FISH) on metaphases generated from peripheral blood lymphocytes revealed only murine chromosomes, also ruling out the possibility of fusion between human and mouse cells. To further characterize these murine leukemias, we performed array CGH on murine spleen gDNA of four immunophenotypically different mice. All mice showed recurrent clonal chromosomal aberrations frequently found in AML. Surprisingly, we have no evidence for the presence of FLT3-ITD in these murine leukemias, suggesting that CD34+FLT3-ITD+ stem cells can trigger development of acute leukemia. We propose that leukemogenesis may mechanistically be related to the host microenvironment and that the bone marrow niche in NOD/SCID mice is susceptible to modulation by the FLT3-ITD oncogene. Disclosures: No relevant conflicts of interest to declare.

Mesodermal Development From Reprogrammed Fanconi Anemia Cells Is Affected by ALDH2 Enzymatic Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 648-648
Author(s):  
Naoya Suzuki ◽  
Asuka Hira ◽  
Akira Niwa ◽  
Megumu Saito ◽  
Keitaro Matsuo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Developmental Stage ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
A Genome ◽  
The Impact ◽  
Mesodermal Development

Abstract Abstract 648 Introduction Fanconi anemia (FA) is a genome instability disorder with clinical characteristics including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. To date 15 genes have been implicated in FA, and their products form a common DNA repair network often referred to as “FA pathway”. Following DNA damage or replication stress, the FA pathway is activated, leading to the monoubiquitination of FANCD2 and FANCI proteins (the ID complex). The monoubiquitinated ID complex is loaded on damaged chromatin with subnuclear foci formation, and mediates homologous recombination. Since cells derived from FA patients are hypersensitive to treatments that induce DNA interstrand cross-links (ICLs), the FA pathway has been considered to function in ICL repair. However, it still remains unclear what type of endogenous DNA damage is repaired through the FA pathway and is the cause of phenotypes in FA patients. Recent studies have suggested that cells deficient in the FA pathway are also sensitive to formaldehyde and acetaldehyde. Aldehydes may create DNA adducts including ICLs or protein DNA crosslinking. These results raise a possibility that the FA pathway prevents BMF by mitigating genotoxicity due to endogenous aldehydes. It has been known that ALDH2 deficiency resulting from Glu487Lys substitution (A allele) is prevalent in East Asian populations. While the Glu487 form (G allele) is proficient in aldehyde catabolism, even the GA heterozygote displayed strongly reduced catalysis because ALDH2 is a tetrameric enzyme and the variant form can suppress the activity in a dominant negative manner. Therefore some Japanese FA patients are expected to be deficient in ALDH2, providing an opportunity to test role of ALDH2 and aldehyde metabolism in human FA patients. Results and discussion In FA fetus, p53/p21 axis has already activated in fetal liver (Ceccaldi, Cell stem cell, 2012), indicating the possibility that hematopoietic defects in FA patients originates from an earlier developmental stage. Since human hematopoietic system originates from embryonic mesoderm, we set out to estimate the role of ALDH2 and FANCA pathway during early embryogenesis. For this, we reprogrammed somatic cells from a patient with ALDH2 GA genotype and observed their in vitro mesodermal differentiation. We first introduced reprogramming factors into fibroblasts by episomal vectors, and obtained colonies which are morphologically compatible with human induced pluripotent stem cells (iPSCs). These iPSC-like cells (designated as FA-iPLCs) showed close similarity to conventional ES/iPSCs regarding marker gene expressions and differentiation ability into three germ layers. We obtained gene-complemented FA-iPLCs (designated as cFA-iPLCs) for control study. To evaluate the impact of ALDH2 activity on iPSC- or iPLC-derived mesodermal differentiation, we next adapted the previously reported serum-free monolayer culture system. Both FA- and cFA-iPLCs showed similar differentiation manners with conventional embryonic stem cells and iPSCs, and percentages of KDR+ mesodermal progenitors including KDR+CD34+ common hemoangiogenic progenitors were comparable. Notably, ALDH2 agonist Alda1 did increase only FA-iPLC-derived mesodermal progenitors but not cFA-iPLCs. These data supported the hypothesis that mesodermal development towards hematopoietic cells in human can be affected by ALDH2 activity in the absence of FA pathway. To confirm the hypothesis, next we set out to assess whether the variation in ALDH2 affects symptoms in Japanese FA patients. Strikingly, we found that progression of BMF was strongly accelerated in heterozygous carrier of the variant A allele compared to homozygous GG patients. Furthermore we looked at occurrence of leukemia and/or myelodysplasia and the somatic developments. Interestingly, these were not significantly difference between patients with each variation of ALDH2, indicating the possibility that aldehydes affect only in early hematopoietic development, not other mesodermal tissues. Overall, our results from FA-iPLCs and clinical study indicate that the variation in ALDH2 affects the occurrence of bone marrow failure in FA patients, and that hematopoietic defect in FA patients is caused by aldehydes in early mesodermal developmental stage. Disclosures: No relevant conflicts of interest to declare.

Elucidating the role of P63 during development of the mammalian nervous systems

2007 ◽  
Vol 30 (4) ◽  
pp. 79
Author(s):  
Sagar Dugani ◽  
Annie Paquin ◽  
David R. Kaplan ◽  
Freda D. Miller
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Cortical Neurons ◽  
Sympathetic Neurons ◽  
Fold Increase ◽  
P53 Family ◽  
Ongoing Work ◽  
Cortical Precursors

Background: The protein p63, a recently discovered member of the p53 family of proteins, is implicated in the maintenance and differentiation of stem cells in the epidermis and is involved in the regulation of naturally-occurring cell death in sympathetic neurons of the peripheral nervous system. Since initial data from our laboratory indicated that p63 is also widely expressed in stem cells and neurons within the developing brain, we assessed its involvement in regulating the genesis and survival of developing cortical neurons. As neurogenesis is initiated at embryonic day 12 (E12), we isolated cortical precursors from p63-/- embryos at E14 and cultured them for 2 days in vitro (DIV). Methods: Based on immunocytochemistry to known markers of apoptosis and neurons, we assessed the level of cell death and neurogenesis. Results: Compared to p63+/+ cortical precursors, p63-/- precursors from littermates showed a 50 % reduction in neuronal death, as assessed by the apoptosis marker, cleaved caspase 3. Interestingly, the proportion of neurons and astrocyte precursors, the latter identified by S100b was also reduced in p63-/- embryos, as compared to p63+/+ littermates. Conclusions: These results suggest that p63 may be involved in the regulation of cell survival and in the differentiation of precursors into neurons and astrocytes. To assess the former, we overexpressed TAp63a, a full-length isoform of p63, in E12/13 cortical precursors and assessed the level of cell death after 2 DIV. Compared to control cells, cells transfected with TAp63a demonstrated a 2-fold increase in cell death. Ongoing work will characterize p63 involvement in differentiation of precursor cells into neurons and astrocytes. To assess if these findings are relevant in vivo, we will use p63flox,flox X Nextin-Cre mice, which have p63 specifically ablated in neural precursors. We will analyze the survival, proliferation, and fate of these p63-/- cells. Together, these studies will help to determine a role for p63 in neural proliferation and apoptosis, processes central to development and response to injury.

Abstract 130: Ox-LDL Impairs The Survival Of Bone Marrow Stem Cells Partially Through Membrane Damage Independent Of ROS Production In Vitro

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Xin Li ◽  
Yuan Xiao ◽  
Yuqi Cui ◽  
Hua Zhu ◽  
Chandrakala A Narasimhulu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Death ◽  
Membrane Damage ◽  
Final Concentration ◽  
Bone Marrow Stem Cells ◽  
Cell Membrane Damage ◽  
Cell Based Therapy

Aims: cell-based therapy with bone marrow stem cells (MSCs) remains a viable option for tissue repair and regeneration. One of the major challenges for cell-based therapy is the limited survival of the cells after in vivo administration. The exact mechanism(s) for impaired in vivo survival of the implanted MSCs remains to be defined. Oxidized low-density lipid protein (ox-LDL) is a natural product in human blood, and the major contributor to the development of atherosclerosis. The present study was to investigate the effect of ox-LDL on the survival of bone marrow stem cells and the mechanisms in vitro. Methods and Results: Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL (with the final concentration of 10 and 20 ug/ml) for up to 48 hours. Exposure to ox-LDL resulted in significant cell death and apoptosis of MAPCs in association with a significant increase in LDH release in the conditioned media in a dose- and time-dependent manner, indicating significant cell membrane damage. The membrane damage was further confirmed with the rapid entry of the small fluorescent dye FM1-43 as detected using confocal microscope. Ox-LDL generated a significant amount of reactive oxygen species (ROS) in the culture system as measured with electron paramagnetic resonance spectroscopy. The antioxidant N-acetylcysteine (NAC, 0.1 mM) completely inhibited the production of ROS from ox-LDL. However, it didn’t prevent ox-LDL-induced cell death or apoptosis. However, pre-treatment of the cells with the specific membrane protective recombinant human MG53 protein (rhMG53)(66 ug/ml, final concentration) significantly, reduced LDH release and the entry of FM1-43 dye into the cells exposed to ox-LDL. Conclusion: Ox-LDL enhanced cell death and apoptosis of MAPCs with a mechanism independent of ROS generation in vitro. Ox-LDL impaired the survival of MAPCs partially through cell membrane damage in vitro.

Activation of AKT1 in Hematopoietic Stem Cells Causes a Myeloproliferative Disease in a Tet-Inducible Mouse Model.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1356-1356
Author(s):  
Christian Brandts ◽  
Miriam Rode ◽  
Beate Lindtner ◽  
Gabriele Koehler ◽  
Steffen Koschmieder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
De Novo ◽  
Myeloproliferative Disorder ◽  
Myeloproliferative Disease ◽  
Hematopoietic Stem ◽  
Akt Phosphorylation

Abstract Activating mutations in Flt3, N- and K-Ras have been reported in all AML subtypes and represent common molecular defects in de novo AML. We have previously shown that these mutations lead to constitutive AKT phosphorylation and activation. As a consequence, Akt phosphorylation is found in myeloid blasts of the majority of AML patients. We reasoned that constitutively active AKT may contribute to leukemia development, and therefore we assessed the contribution of AKT in oncogenic transformation in vivo. For this purpose, we established an inducible mouse model expressing myristylated AKT1 under the control of the scl-3′ enhancer (MyrAKT1). This system restricts activated AKT1 to endothelium, hematopoietic stem cells and myeloid lineage cells at a low but detectable level. About 40% of induced mice developed a myeloproliferative disorder after latencies of 7 to 22 months. Onset of disease was frequently associated with hemangioma formation, due to endothelial MyrAKT1 expression. The myeloproliferative disorder was associated with splenomegaly with increased extramedullary hematopoiesis, while the peripheral blood contained mature granulocytes. Furthermore, the stem cell and progenitor cell compartment in spleens and bone marrow of these mice was altered compared to control mice. Colony formation assays with MyrAKT1-expressing bone marrow suggested that overactivation of AKT1 enhanced proliferation. The AKT1-induced disease was transplantable by both bone marrow and spleen cells. These findings highlight the oncogenic capacity of constitutively activated AKT1 in vivo and indicate that AKT is an attractive target for therapeutic intervention in AML.

Growth Factor Independence 1 (Gfi1) Is An Essential Factor for the Development of Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 297-297
Author(s):  
Cyrus Khandanpour ◽  
Ehssan Sharif- Askari ◽  
Paul Jolicoeur ◽  
Ulrich Duehrsen ◽  
Tarik Moroy
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Cell Death ◽  
T Cell ◽  
Cell Lymphoma ◽  
Latency Period ◽  
T Cell Lymphoma ◽  
Hematopoietic Stem

Abstract Hematopoietic differentiation is controlled to a large extent by a network of transcription factors and chromatin modifiers and disruption of this system can lead to leukemia or lymphoma. One of the transcription factor genes, which is aberrantly expressed in human T-cell lymphoma is Growth Factor Independence 1 (Gfi1). Since over expression of Gfi1 can accelerate experimentally induced T-cell tumors in mice, it is likely that Gfi1 plays a crucial role in establishing or maintaining lymphoid neoplasms. To test this hypothesis we have used, N-ethyl-N-nitrosourea (ENU) to induce T-cell tumors in WT mice (Gfi1+/+), Gfi1-deficient mice (Gfi1−/−) or mice transgenically over expressing Gfi1 under the control of the pan-hematopoietic vav-promoter (vav-Gfi1). As expected, most of Gfi1+/+ mice (25/27) developed T-cell tumors and acute myeloid leukemia within 118 days. Similarly, vav-Gfi1 mice (10/10) developed T-cell lymphoma, but within a shorter latency period (88 days). In contrast, only 3/14 Gfi1−/− mice developed hematopoietic neoplasia with a prolonged median latency period of 126 days. Other approaches using infection of newborn mice with Moloney Murine leukemia virus (MoMuLV) to induce T-cell lymphoma or co expression of an Eμ-myc transgene to induce B-cell lymphoma showed a similar dependency of tumor formation on the presence and expression of Gfi1. Closer analysis of tumors forming in Gfi1−/− mice demonstrated that Gfi1 deficiency correlated with a smaller size of the tumors and a noticeably increased rate of cell death within the tumor samples. This pointed to a potential role of Gfi1 in the regulation of apoptosis. To explore this hypothesis, we exposed both thymocytes and hematopoietic stem cells (Lin-, Sca1+, c-kit+, LSK) to ENU or gamma-irradiation in vitro. We could observe that Gfi1−/− thymocytes and stem cells (LSK cells) have a higher rate of cell death following exposure to these DNA damage inducing agents in vitro than the WT controls. To validate these results, we recapitulated these experiments in vivo. Gfi1−/− mice exhibited severe bone marrow failure and a more pronounced loss of hematopoietic stem cells (LSK) than Gfi1+/+ mice after ENU treatment or gamma irradiation in vivo. To explore this mechanism on the molecular basis we evaluated expression of the different pro and antiapoptotic components in Gfi1+/+ and Gfi1−/− thymocytes after irradiation. Strikingly, Gfi1−/− thymocytes expressed higher levels of the pro-apoptotic proteins such as Bax and Noxa and lower levels of the CDK inhibitor p21WAF than WT thymocytes following induction of DNA damage. Our model would be that Gfi1 represents a new regulator in the cellular response to DNA damage in the hematopoietic system by inhibiting different proapoptotic factors. We propose that Gfi1 is essential for the development of lymphoid and potentially myeloid neoplasms by inhibiting apoptosis. We suggest that Gfi1 could represent a possible new target structure for therapeutic intervention.

In Vivo Selection and Long-Term Engraftment Of Hematopoietic Stem Cells Generated Via Vascular Niche Induction Of Nonhuman Primate Induced Pluripotent Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 466-466
Author(s):  
Jennifer L Gori ◽  
Jason M Butler ◽  
Devikha Chandrasekaran ◽  
Brian C Beard ◽  
Daniel J Nolan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Nonhuman Primate ◽  
Fold Increase ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P<0.05). On average, 15% CD34+ and 37% CD45+ cells were detected in the bone marrow of mice transplanted with E4ORF1+EC-expanded P140K-MniPSC HSCs, which is significantly higher than levels detected in the other cohorts (Table 1). CD45+ cells in the marrow were predominantly myeloid but lymphoid subsets were also present (10-25% CD3+ cells). Remarkably, the level of gene marking in CFCs and number of gene marked CFCs from mouse bone marrow was substantially higher for mice transplanted with E4ORF1+EC expanded compared to cytokine expanded P140K-MniPSC-HSCs (Table 1). Finally, to confirm engraftment of authentic HSCs, secondary transplants were established. Although engraftment was achieved in all secondary transplanted cohorts, the level of nonhuman primate cells detected was significantly higher in animals transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs. Significantly more lymphocytes (CD45+CD3+ and CD45+CD56+) and monocytes (CD45+CD14+) were detected in the blood of these secondary transplant recipients. These findings confirm generation of bona fide HSCs derived from nonhuman primate iPSCs and demonstrate that O6BG/BCNU chemotherapy supports in vivo selection of P140K-MniPSC-HSCs generated by co-culture with the E4ORF1+EC vascular platform. Our studies mark a significant advance toward clinical translation of PSC-based blood therapeutics and the development of a nonhuman primate preclinical model. Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0 Disclosures: Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.

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