scholarly journals Cell Cycle Status of CD34+ Hemopoietic Stem Cells Determines Lentiviral Integration in Actively Transcribed and Development-related Genes

2015 ◽  
Vol 23 (4) ◽  
pp. 683-696 ◽  
Author(s):  
Eleni Papanikolaou ◽  
Anna Paruzynski ◽  
Ioannis Kasampalidis ◽  
Annette Deichmann ◽  
Evangelos Stamateris ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hemopoietic Stem Cells ◽  
Lentiviral Integration ◽  
Cell Cycle Status

Hematopoiesis in the Elderly: Age-Associated Effects in Frequency, Function, and Gene Expression of Human Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1505-1505
Author(s):  
Wendy W. Pang ◽  
Elizabeth A. Price ◽  
Irving L. Weissman ◽  
Stanley L. Schrier
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Expression Profiles ◽  
Frequency Function ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cell Cycle Status

Abstract Abstract 1505 Poster Board I-528 Aging of the human hematopoietic system is associated with an increase in the development of anemia, myeloid malignancies, and decreased adaptive immune function. While the hematopoietic stem cell (HSC) population in mouse has been shown to change both quantitatively as well as functionally with age, age-associated alterations in the human HSC and progenitor cell populations have not been characterized. In order to elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated and compared HSC and other hematopoietic progenitor populations prospectively isolated via fluorescence activated cell sorting (FACS) from 10 healthy young (20-35 years of age) and 8 healthy elderly (65+ years of age) human bone marrow samples. Bone marrow was obtained from hematologically normal young and old volunteers, under a protocol approved by the Stanford Institutional Review Board. We determined by flow cytometry the distribution frequencies and cell cycle status of HSC and progenitor populations. We also analyzed the in vitro function and generated gene expression profiles of the sorted HSC and progenitor populations. We found that bone marrow samples obtained from normal elderly adults contain ∼2-3 times the frequency of immunophenotypic HSC (Lin-CD34+CD38-CD90+) compared to bone marrow obtained from normal young adults (p < 0.02). Furthermore, upon evaluation of cell cycle status using RNA (Pyronin-Y) and DNA (Hoechst 33342) dyes, we observed that a greater percentage of HSC from young bone marrow are in the quiescent G0- phase of the cell cycle compared to elderly HSC, of which there is a greater percentage in G1-, S-, G2-, or M-phases of the cell cycle (2.5-fold difference; p < 0.03). In contrast to the increase in HSC frequency, we did not detect any significant differences in the frequency of the earliest immunophenotypic common myeloid progenitors (CMP; Lin-CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP; Lin-CD34+CD38+CD123+CD45RA+), and megakaryocytic-erythroid progenitors (MEP; Lin-CD34+CD38+CD123-CD45RA-) from young and elderly bone marrow. We next analyzed the ability of young and elderly HSC to differentiate into myeloid and lymphoid lineages in vitro. We found that elderly HSC exhibit diminished capacity to differentiate into lymphoid B-lineage cells in the AC6.21 culture environment. We did not, however, observe significant differences in the ability of young and elderly HSC to form myeloid and erythroid colonies in methylcellulose culture, indicating that myelo-erythroid differentiation capacity is preserved in elderly HSC. Correspondingly, gene expression profiling of young and elderly human HSC indicate that elderly HSC have up-regulation of genes that specify myelo-erythroid fate and function and down-regulation of genes associated with lymphopoiesis. Additionally, elderly HSC exhibit increased levels of transcripts associated with transcription, active cell-cycle, cell growth and proliferation, and cell death. These data suggest that hematopoietic aging is associated with intrinsic changes in the gene expression of human HSC that reflect the quantitative and functional alterations of HSC seen in elderly bone marrow. In aged individuals, HSC are more numerous and, as a population, are more myeloid biased than young HSC, which are more balanced in lymphoid and myeloid potential. We are currently investigating the causes of and mechanisms behind these highly specific age-associated changes in human HSC. Disclosures: Weissman: Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: ; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

scholarly journals Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells

1993 ◽  
Vol 122 (4) ◽  
pp. 897-902 ◽  
Author(s):  
WH Fleming ◽  
EJ Alpern ◽  
N Uchida ◽  
K Ikuta ◽  
GJ Spangrude ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Significant Proportion ◽  
Cell Activity ◽  
Rhodamine 123 ◽  
Hematopoietic Stem ◽  
Cell Cycle Status

Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic cell lineages while retaining their capacity for self renewal. One of the predictions of this model is the existence of a heterogeneous pool of HSCs, some members of which are destined to become lineage restricted progenitor cells while others function to renew the stem cell pool. To test whether HSCs are heterogeneous with respect to cell cycle status, we determined the fraction of phenotypically defined murine HSCs (Thy1.1lo Lin-/lo Sca-1+) that contain &gt; 2n amount of DNA as measured by propidium iodide staining, Hoechst dye uptake and [3H]thymidine labeling; that fraction is 18-22%. In contrast, in the developing fetal liver, 40% of HSCs are in the S/G2/M phases of the cell cycle. Those HSCs which exhibit a low level of staining with rhodamine 123 are almost exclusively in G0/G1 (97%) whereas only 70% of HSCs which stain brightly for rhodamine 123 are in G0/G1. The injection of 100 G0/G1 HSCs rescued 90% of lethally irradiated mice in contrast to 100 S/G2/M HSCs, which protected only 25% of lethally irradiated recipients. Enhanced long-term donor-derived multilineage reconstitution of the peripheral blood was observed in recipients of 100 G0/G1 HSCs compared to recipients of 100 S/G2/M cells. These data indicate that a significant proportion of HSCs are actively proliferating during steady state hematopoiesis and that this subpopulation of cells exhibits reduced stem cell activity.

scholarly journals Hydroxyurea Can Be Used to Increase Mouse c-kit+Thy-1.1loLin−/loSca-1+ Hematopoietic Cell Number and Frequency in Cell Cycle In Vivo

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4354-4362 ◽  
Author(s):  
Nobuko Uchida ◽  
Annabelle M. Friera ◽  
Dongping He ◽  
Michael J. Reitsma ◽  
Ann S. Tsukamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Cell Number ◽  
Short Term ◽  
Synthesis Inhibitor ◽  
Hematopoietic Stem ◽  
Cell Cycle Status

Abstract The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1loLin−/loSca-1+ (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

Cell Cycle Entry Potentiates Elimination of Chemotherapy-Resistant Human AML Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1422-1422
Author(s):  
Yoriko Saito ◽  
Naoyuki Uchida ◽  
Satoshi Tanaka ◽  
Mariko Tomizawa-Murasawa ◽  
Nahoko Suzuki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Caspase 3 ◽  
T Test ◽  
Tunel Staining ◽  
Cell Cycle Entry ◽  
Cell Cycle Status ◽  
Active Caspase

Abstract Abstract 1422 Poster Board I-445 Acute myeloid leukemia (AML) is associated with poor long-term prognosis despite advances in therapeutic modalities over the past few decades. As leukemia stem cells (LSCs) capable of AML initiation may contribute to recurrent disease, LSC-targeted therapies are required to overcome disease relapse and to improve long-term patient outcomes. We previously reported that human AML CD34+CD38- cells self-renew, generate non-stem leukemic cells, and possess potential to initiate leukemia following engraftment of newborn NOD/SCID/IL2rgKO mice. In the recipient bone marrow (BM), AML LSCs were found to reside preferentially within the endosteal region and exhibited chemotherapy resistance. In addition, we observed that AML cells abutting the BM endosteum were cell cycle quiescent while AML cells in the center of the BM were cycling. Based on these findings, we hypothesized that induction of cell cycle entry in quiescent AML LSCs may increase their susceptibility to chemotherapeutic agents, leading to enhanced elimination of LSCs. To test this hypothesis, we assessed the effect of granulocyte colony-stimulating factor (G-CSF) on cell cycle status and chemotherapy susceptibility of primary human AML LSCs in vivo using the NOD/SCID/IL2rgKO xenotransplantation model. In AML-engrafted recipient mice transplanted with LSCs from seven AML patients, flow cytometric analyses demonstrated a significant reduction of quiescent LSCs following 300μg/kg G-CSF sc daily for 5 days (%G0 within hCD34+CD38- BM cells (mean+/-s.e.m): 49.2+/-2.6 (n=47) and 20.5+/-2.0 (n=36), control and G-CSF treated recipients, respectively, p<0.0001 by two-tailed t test). Direct examination of recipient BM in situ revealed cell cycle entry of human AML cells abutting the BM endosteum as evidenced by increased Ki67 expression. Next we developed an in vivo treatment model evaluating the effect of cell cycle induction on chemotherapy-responsiveness of human primary AML LSCs. Human AML-engrafted recipients received AraC alone (1g/kg ip daily for 2 days) or G-CSF followed by AraC (300μg/kg G-CSF sc daily for 5 days with 1g/kg AraC ip daily on days 4 and 5). The proportion of viable active caspase 3-negative human LSCs decreased significantly with pre-chemotherapy cell cycle induction (% active caspase 3-negative hCD34+CD38- BM cell (mean+/-s.e.m.): 82.7+/-1.3% (n=33) and 40.4+/- 3.1% (n=30), AraC alone- and G-CSF followed by AraC-treated recipients, respectively, p<0.0001 by two-tailed t test). TUNEL staining of the recipient BM showed increased apoptosis of AML cells abutting the BM endosteum in recipients receiving AraC following cell cycle induction. Limiting dilution serial transplantation of residual viable human AML cells in the BM of treated recipients showed 100-fold reduction in the frequency of LSCs capable of initiating AML in secondary recipients (BM LSC frequency: 1/560 (n=125) and 1/55,076 (n=109), AraC alone- and G-CSF then AraC-treated recipients, respectively, p=0.0001 by two-tailed t test). At 24 weeks post-transplantation, 89.4% of secondary recipients of G-CSF followed by AraC-treated mice survived compared with only 2.0% survival in secondary recipients of AraC alone-treated mice (p<0.0001, survival estimated by Kaplan-Meier method). These findings indicate that cell cycle status is a key determinant of LSC chemo-responsiveness and that therapeutic strategies promoting LSC cell cycle entry may improve outcomes in AML. Disclosures: No relevant conflicts of interest to declare.

Polycomb group gene mel-18 modulates the self-renewal activity and cell cycle status of hematopoietic stem cells

2004 ◽  
Vol 32 (6) ◽  
pp. 571-578 ◽  
Author(s):  
Teruyuki Kajiume ◽  
Yuichi Ninomiya ◽  
Hiroto Ishihara ◽  
Rieko Kanno ◽  
Masamoto Kanno
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Polycomb Group ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Cycle Status ◽  
Polycomb Group Gene

907 Fasting Alters Cell Cycle Status in Both Rapidly and Slowly Cycling Intestinal Stem Cells

2012 ◽  
Vol 142 (5) ◽  
pp. S-159
Author(s):  
Camilla A. Richmond ◽  
Bristol Brandt ◽  
Diana L. Carlone ◽  
Robert K. Montgomery ◽  
David T. Breault
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Intestinal Stem Cells ◽  
Cell Cycle Status

Expansion and Multipotencial Differentiation of Mesenchymal Stem Cells Isolated from Patients after High Dose Chemotherapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4259-4259
Author(s):  
Karen L. Prata ◽  
Maristela D. Orellana ◽  
Aparecida M. Fontes ◽  
Karina R. Solano ◽  
Simone Kashima ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
High Dose Chemotherapy ◽  
High Dose ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Cell Cycle Status ◽  
Supportive Function

Abstract Background. High dose chemotherapy (HDCT) followed by autologous PBSC rescue has been increasingly used for the treatment of several human diseases. However, little is known on the extent of this therapy on the marrow mesenchymal stem cells (MSCs). Aims. To evaluate the feasibility of expansion and multipotencial differentiation of MSCs isolated from patients after HDCT. Patients and Methods. Twelve lymphoma’s patients (LP) free of disease in bone marrow (BM) were enrolled in the study. They were submitted to BEAM’s protocol with autologous PBSC rescue 28 to 1836 days before the sample collection. Six normal bone marrow donors (ND) were used as controls. The LP and ND median age were 37.5 (range 22–49) and 31.5 years old (range 23–42), respectively. MSCs were isolated by plastic adherence and expanded ex vivo by cultivation in flasks with α-MEM with 15% fetal bovine serum. Media was changed every 3–4 days. At 90% confluence, the cells were re-plated and expanded. The isolation efficiency, colony-forming unit-fibroblast (CFU-F) frequency, growth kinetics, phenotypic characteristics, cell cycle status, multi-lineage differentiation capacity as well as hematopoiesis-supportive function were determined and compared with those of ND-MSCs. This study protocol and the consent form were approved by the institution ethics committees. Results. The results were analyzed by Mann-Whitney test and are expressed as median (range) to LP and ND, respectively. MSCs were successful isolated from all BM samples collected for this study. The cell population showed typical fibroblast-like morphology, appearing as an adherent, spindle shaped cell layer and growing to confluence after a few weeks of culture. The number of CFU-F found at 14 days of culture were 0.94 (0.00–3.75) and 1.25 (0.13–9.25) x10−5 nucleated cells (p = 0.4421). The doubling time between the 1st and 2nd passages was 80.66 (34.08–195.35) and 46.30 (36.36–270.59) hours (p = 0.1025). The cell clones proliferated extensively until 8.17 (1.81–28.27) and 18.11 (11.85–27.48) population doublings (p = 0.0668) in 71.50 (46–88) and 81 (57–103) cultivation days (p = 0.1505). Immunophenotypically, these cells were positive for the CD73, CD105, CD90, CD29, CD13, CD44, CD49e, CD54, HLA-class 1 and Stro-1 markers and negative for CD34, CD45, CD14, CD51/61, HLA-DR and KDR. Regarding the cell cycle status, 85.63 (63.19–92.17) and 82.41 (82.19–87.02) % were in GO-G1 phase (p = 1,000), while only 12.17 (3.33–36.81) and 10.67 (6.59–12.05) % were in S phase (p = 0,6828). All samples tested were capable of differentiating along adipogenic, osteogenic and chondrogenic lineages in vitro, demonstrated by morphology, cyto- and imunohistochemistry or RT-PCR reaction (PPARg and osteopontin genes expression). After co-culture with CD34+ cord blood cells for 1 and 4 weeks, no significant difference CD34+ expansion or colony-forming cells (BFU-E or CFU-GM) were observed between the CD34+ cells/LP-MSCs and CD34+ cells/ND-MSCs co-cultures with cytokines or not. Interpretation and Conclusions. Our results demonstrate that is possible to cultivate and expand MSCs with multipotential differentiation capabilities and hematopoiesis-supportive function from patients after HDCT. Despite there were no significant differences in the median values between LP and ND, the comparative study indicates a possible damage in MSCs by HDCT.

A General Theory of Marrow Stem Cell Fate Determination

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4794-4794
Author(s):  
Peter J Quesenberry ◽  
Mark S Dooner ◽  
Laura R Goldberg
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Tritiated Thymidine ◽  
Short Exposure ◽  
Stem Cell Population ◽  
Hoechst 33342 ◽  
Cell Cycle Status ◽  
Marrow Stem Cell

Abstract Abstract 4794 We propose that the long-term multipotent marrow renewal stem cell is on a continuum of change in potential tied to cell cycle stage. Furthermore our data indicate that marrow stem cells cannot be defined on a clonal basis and that purification of stem cells, as is currently carried out, leads to a biased population which provides only limited information with regard to marrow stem cells. Previous data have clearly demonstrated continuous and reversible phenotype change when murine marrow stem cells are driven through cell cycle in vitro by cytokines (Quesenberry et al, Exp Hematol, 2010). The relevance of this to normal hematopoiesis is placed into question by numerous observations indicating that highly purified long-term hematopoietic stem cells are in the G0 phase of cell cycle. However, no studies have addressed the cell cycle status of long-term multilineage stem cells in vivo from normal unseparated marrow. Given the fact that marrow stem cell separations are not random and that over 90% of stem cells are lost in these separations (Nilsson et al, Blood, 1997), the possibility exists that the purified stem cells are not representative of stem cells in the whole population. Accordingly, we have investigated the cell cycle status of murine whole marrow stem cells employing Hoechst 33342 alone or pyronin/Hoechst 33342 staining with FACS separation into G0, G1 and S/G2/M fractions, followed by competitive engraftment of each sub fraction into lethally irradiated mice and analysis of multilineage engraftment from 28 to 52 weeks. In five separate experiments we have found that 50 ± 4 % of whole marrow long-term engrafting cells were in S/G2/M. Engraftment in every instance was multilineage as determined by analysis of B220, Gr-1. CD11b and CD3 engraftment. Given that this is an instantaneous look at cell cycle status, it implies that virtually all long-term engrafting cells are in active cell cycle. Similar studies on LT-HSC (lin-Sca+c-kit+ Flk2-) essentially confirmed previous studies (with one stochastic exception) that LT-HSC are a dormant, noncycling population in G0. In order to further confirm these studies, donor whole marrow was subjected to high-specific activity tritiated thymidine suicide for thirty minutes and long-term multilineage engraftment was determined in lethally irradiated mice. In these on-going studies we find that 52 ± 8.6 % of engrafting cells were in S/G2/M out to 6 months (5 experiments). Given the short exposure to tritiated thymidine, these data also indicate that virtually all marrow stem cells are in cell cycle. With progression through cell cycle there is a continuous change in all aspects of cellular phenotype and thus the characteristics of the stem cell will vary from moment to moment. In this setting, purification of individual cells can not define the population. Thus the marrow long-term repopulating stem cell is on a continuum of change in potential which will be realized if interrogated with appropriate stimuli. These potentials continuously change with progression through cell cycle with different fate outcomes at different points in cell cycle. The present challenge for the field is to appropriately define the stem cell population, not the “stem cell”. Disclosures: No relevant conflicts of interest to declare.

Junctional Adhesion Molecule 2 Represents a Novel Subset of Hematopoietic Stem Cells Poised for T Lymphopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3862-3862 ◽  
Author(s):  
Visnja Radulovic ◽  
Mark van der Garde ◽  
Valgardur Sigurdsson ◽  
Alya Zriwil ◽  
Svetlana Soboleva ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Cell Cycle Status

Abstract Phenotypically well-characterized hematopoietic stem cells (HSCs) still represent a heterogeneous pool of primitive cells regarding to their functionality. In particular, different lineage potential of HSCs have been considered as one of key features of the HSC heterogeneity. The lineage output of HSCs is often coupled with cell cycle status or long-term reconstitution potential, however molecular mechanisms of the mutuality are unclear and other type of the regulation may exist. In addition, prospective isolation of such HSCs biased towards specific lineage(s) is still problematic, as many of categorizations highly rely on retrospective information, e.g. transplantation assay. Although several markers have been reported to be able to subdivide HSCs into subcategories, exploration of additional markers will allow us understanding further molecular mechanisms of HSC regulations including activation and lineage choice. Here, we show that cell surface expression of Junctional adhesion molecule 2 (Jam2) represents higher reconstitution capacity of HSCs and the T cell potential. Flow cytometry analyses revealed that a subset of CD150+CD48-KSL cells in mouse bone marrow (BM) were positive for Jam2 (Jam2+HSC, 36.6 ±13.0 %), while other Jam family member Jam1 (F11r) was expressed on all HSCs and Jam3 was not detected. To examine functional differences of Jam2+ and Jam2-HSCs, 30 cells were separately transplanted into lethally irradiated mice. Peripheral blood analyses revealed that Jam2+HSCs reconstituted more efficiently than Jam2-HSCs (77.5 ±15.9 and 51.7 ±29.3 %, respectively). In case of transplantation using 5 cells, the frequency of reconstituted mice was higher in Jam2+HSCs (7 in 11) compared to Jam2-HSCs (4 in 11), indicating that Jam2+ population is more enriched for functional HSCs. The expression of Jam2 on HSC is reversible, but not hierarchical, as both Jam2+ and Jam2-HSCs reconstituted opposite population in the BM.Lineage analyses revealed that Jam2+HSCs have a greater potential in lymphoid cell reconstitution, particularly T cells, whereas the chimerism in myeloid cells was not significantly different from Jam2-HSCs. This tendency of higher contribution to the T cell development was even more pronounced in the secondary transplantation experiments, where the contribution of Jam2+HSCs in T cells was close to 100 %. Of note, most of Jam2+HSCs were in a dormant state, suggesting that the T cell (or lymphoid) potential of Jam2+HSCs is independent of cell cycle progression. Jam2 has been reported to interact with Jam1, which mediates the Notch signaling (Kobayashi et al., Nature, 2014). Competitive co-culture of Jam2+ vs Jam2-HSCs on OP9-DL1 showed that Jam2+HSCs dominated the T cell production, whereas no difference was seen in B cell production upon OP9 co-culture. Since Jam2 positivity correlates to T cell potential, we asked if altered T lymphopoiesis environment affects the cell surface Jam2 expression. Comparison of C57BL/6, NOD, NOD-Scid and NOD-Scid Il2rγ KO (NSG) mice showed that HSCs of NSG mice have significantly higher frequency of Jam2+HSCs, suggesting that cell surface Jam2 expression might be regulated by specific cytokine(s) binding to IL2Rγ. Our findings suggest Jam2 is a new marker for a subset of HSCs that preferentially generate T cells. In addition, this work uncouples the lineage choice and cell cycle status, which proposes a novel model to the lineage-determining machineries. Since efficient and immediate generation of T cells in transplantation therapy is important to minimize infectious risks, understanding the molecular basis of the Jam-Notch cooperation would contribute to establish safer and more efficient treatment. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document