TIMP-1 Deficiency Subverts Cell Cycle Dynamics In HSCs.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1545-1545
Author(s):  
Lara Rossi ◽  
Margaret A. Goodell
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Biological Role ◽  
Biological Behavior ◽  
Hematopoietic Stem ◽  
Cell Cycle Inhibitors ◽  
Cell Cycle Dynamics

Abstract Abstract 1545 Although TIMP-1 was initially described as a mere inhibitor of Metalloproteinases (MMPs), recent findings have offered a different perspective on its biological role, contributing to unveil its multifaceted nature. In addition to inhibiting MMP activity, TIMP-1 has been proven to play MMP-independent, cytokine-like activities and to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. Bone marrow hematopoietic stem cells (HSCs) were isolated from TIMP-1-/- mice based on the phenotype Side Population c-Kit+Lin- Sca-1+ (SPKLS). In vitro cultural assays as well as in vivo transplantation assays were employed to investigate how TIMP-1 obliteration affects murine hematopoiesis. Cell-cycle dynamics in KO SPKLS HSCs were characterized by Pyronin Y/Hoechst staining, Ki-67 staining, as well as evaluation of RNA expression of cell cycle inhibitors, such as p53, p57, and p21. We found that TIMP-1-/- mice have decreased HSC numbers and, consistent with this finding, TIMP-1-/- HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1-/- LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Of note, TIMP-1-/- HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Interestingly, p53 RNA levels are indeed increased in TIMP-1-/- SPKLS HSCs compared to controls. Likewise, the expression level of other cell-cycle inhibitors, such as p57 and p21, were found to be higher in KO SPKLS HSCs, indicating a disregulation of cell-cycle dynamics.Our study highlights a novel biological role of TIMP-1 in the regulation of the HSC compartment and suggest a novel mechanism presiding over stem cell quiescence in the framework of the BM milieu. Disclosures: No relevant conflicts of interest to declare.

Metabolic Regulation of Hematopoietic Stem Cells During Stress

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-42-SCI-42
Author(s):  
Toshio Suda
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Metabolic Regulation ◽  
Conflicts Of Interest ◽  
Hypoxia Inducible Factor ◽  
Ros Generation ◽  
Hematopoietic Stem

Abstract Abstract SCI-42 Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed. Disclosures: No relevant conflicts of interest to declare.

Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4185-4193 ◽  
Author(s):  
Hanno Glimm ◽  
IL-Hoan Oh ◽  
Connie J. Eaves
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Cell Activity ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
A Cell

Abstract An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G1fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G0/G1and S/G2/M. Interestingly, the G0 cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle–associated change in in vivo stem cell homing, the cultured G0/G1 and S/G2/M CD34+ CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor β1 that increased the G0/G1 fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle–associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo–manipulated grafts.

Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 861-861 ◽  
Author(s):  
Viktor Janzen ◽  
Heather E. Fleming ◽  
Michael T. Waring ◽  
Craig D. Milne ◽  
David T. Scadden
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

Geminin Regulates Hematopoietic Stem Cell Proliferation and Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1478-1478
Author(s):  
Kathryn M. Shinnick ◽  
Kelly A. Barry ◽  
Elizabeth A. Eklund ◽  
Thomas J. McGarry
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Transcription Factors ◽  
Cell Division ◽  
Dna Replication ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Regulatory Protein ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation

Abstract Abstract 1478 Poster Board I-501 Hematopoietic stem cells supply the circulation with mature blood cells throughout life. Progenitor cell division and differentiation must be carefully balanced in order to supply the proper numbers and proportions of mature cells. The mechanisms that control the choice between continued cell division and terminal differentiation are incompletely understood. The unstable regulatory protein Geminin is thought to maintain cells in an undifferentiated state while they proliferate. Geminin is a bi-functional protein. It limits the extent of DNA replication to one round per cell cycle by binding and inhibiting the essential replication factor Cdt1. Loss of Geminin leads to replication abnormalities that activate the DNA replication checkpoint and the Fanconi Anemia (FA) pathway. Geminin also influences patterns of cell differentiation by interacting with Homeobox (Hox) transcription factors and chromatin remodeling proteins. To examine how Geminin affects the proliferation and differentiation of hematopoietic stem cells, we created a mouse strain in which Geminin is deleted from the proliferating cells of the bone marrow. Geminin deletion has profound effects on all three hematopoietic lineages. The production of mature erythrocytes and leukocytes is drastically reduced and the animals become anemic and neutropenic. In contrast, the population of megakaryocytes is dramatically expanded and the animals develop thrombocytosis. Interestingly, the number of c-Kit+ Sca1+ Lin- (KSL) stem cells is maintained, at least in the short term. Myeloid colony forming cells are also preserved, but the colonies that grow are smaller. We conclude that Geminin deletion causes a maturation arrest in some lineages and directs cells down some differentiation pathways at the expense of others. We are now testing how Geminin loss affects cell cycle checkpoint pathways, whether Geminin regulates hematopoietic transcription factors, and whether Geminin deficient cells give rise to leukemias or lymphomas. Disclosures: No relevant conflicts of interest to declare.

Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2504-2504
Author(s):  
Russell Garrett ◽  
Gerd Bungartz ◽  
Alevtina Domashenko ◽  
Stephen G. Emerson
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Hematopoietic Stem ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

Bcr-Abl Impairs T Cell Development From Murine Induced Pluripotent Stem Cells and Hematopoietic Stem Cells; A Partial Explanation for the Concept That Ph+ Clone Never Involves T Cell Lineage in CML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3748-3748
Author(s):  
Bidisha Chanda ◽  
Kiyoko Izawa ◽  
Ratanakanit Harnprasopwat ◽  
Keisuke Takahashi ◽  
Seiichiro Kobayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
T Cell Development ◽  
Cell Lineage ◽  
Cell Development ◽  
Hematopoietic Stem

Abstract Abstract 3748 Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder generally believed to originate from a hematopoietic stem cell carrying the BCR-ABL fusion gene, which generally encodes 210kD and 190kD constitutively active tyrosine kinases termed as p210 and p190, respectively. In spite of the putative stem cell origin and the competence for differentiation toward mature B cells, there is a longstanding consensus that CML never involves the T cell lineage at least in chronic phase. To gain insight into this apparent conflict, we used in vitro T cell differentiation model from murine pluripotent stem cells (PSCs) as well as hematopoietic stem cells (HSCs). C57BL/6 MEFs were reprogrammed using a polycistronic lentiviral Tet-On vector encoding human Oct4, Sox2 and Klf4, which were tandemly linked via porcine teschovirus-1 2A peptides, together with another lentiviral vector expressing rtTA driven by the EF-1a promoter. Almost all the vector sequences including the transgenes were deleted by adenovirus-mediated transduction of Crerecombinase after derivation of iPSCs, and only remnant 291-bp LTRs containing a single loxP site remained in the genome. A clone of MEF-iPSCs were retrovirally transduced with p190DccER, a ligand-controllable p190-estrogen receptor fusion protein, whose tyrosine kinase activity absolutely depends on 4-hydroxytamoxyfen (4-HT).For T cell lineage differentiation, p190DccER-MEF-iPSCs were recovered from a feeder-free culture supplemented with LIF and plated onto a subconfluent OP9-DL1 monolayer in the presence of Flt3 ligand and IL7 with or without 0.5 mM 4-HT.After 3 weeks of culture, iPSC-derived blood cells were collected and subjected to FACS analysis for their lineage confirmation. About 70% of lymphocyte-like cells from the 4-HT(-) culture expressed CD3, but only 20% of counterparts from the 4-HT(+)culture expressed CD3, suggesting impaired T cell development by Bcr-Abl. Next, c-Kit+Sca1+Lin− (KSL) bone marrow cells were prepared by FACS from 8-weeks old C57BL/6 mice treated with 5-FU. KSL cells were similarly transduced with p190DccER and were subjected to the OP9-DL1co-culture system with or without 0.5 mM 4-HT.After 2 weeks of culture, 90% of lymphocytes from the 4-HT(-)culture revealed CD3+TCRβ+ phenotype, but only 30% of those were double positive in the presence of 4-HT(+). In addition, 96% of lymphocytes from the 4-HT(-) culture progressed to the DN2 stage with c-Kit−CD44+CD25+phenotype, whereas 40% of those from the 4-HT(+) culture arrested at the DN1 stage showing c-Kit+CD44+CD25−.Since IL7 plays a central role at the stage from DN1 to DN2 of progenitor T cells, Bcr-Abl is suggested to impair T cell development possibly through interfering with the IL7 signal. The precise mechanism underlying impaired T lymphopoiesis by Bcr-Abl is under investigation. Disclosures: No relevant conflicts of interest to declare.

Periostin Acts As An Important Cell Cycle Regulator Of Adult Hematopoietic Stem Cells Via Binding To Integrin-αvβ3

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 341-341 ◽  
Author(s):  
Satish Khurana ◽  
Catherine M. Verfaillie
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Integrin Αvβ3 ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cell Cycle Regulator ◽  
Blocking Antibodies

Osteoblasts are one of the important cellular components of the niche for hematopoietic stem cells (HSCs) in mammalian bone marrow (BM). Integrin receptors not only play a key role in HSC adhesion within the BM niche but also transfer regulatory signals from the microenvironment to HSCs. Periostin (Postn or osteoblast specific factor-1; OSF-1) is expressed in osteoblasts in addition to many other tissues, and acts as a ligand for Integrin-αvβ3 (ITGAV-B3). We identified POSTN as an important regulator of the cell cycle in adult murine HSCs. POSTN inhibited culture induced proliferation of HSCs thereby decreasing the total number of cells following 2-5 day culture of primitive HSCs, identified as CD150+CD48-Lin-Sca-1+c-kit+ (CD150 KLS) cells with SCF and TPO, while increasing the proportion of long-term (LT-) HSCs. Culture for 5 days with POSTN decreased the short-term (ST-) engraftment of progeny of 200 CD150 KLS cells, while significantly increasing LT- engraftment of the donor derived cells. A significant fraction of CD150 KLS cells expressed ITGAV as well as ITGB3. POSTN did not affect proliferation of HSCs in vitro following blocking of ITGAV with neutralizing antibodies. Among the important cell cycle regulators, we found an increase in p27kip1 expression in HSCs. Preliminary studies on possible signaling mechanisms involved, showed that POSTN inhibits Akt phosphorylation, known to mediate inhibition of both expression and activation of p27Kip1. Intravenous infusion of recombinant POSTN protein significantly decreased proliferation of hematopoietic progenitors as shown by Brdu incorporation and Hoechst/Pyronin staining. Interestingly, POSTN infusion also led to an increase in the number of KLS as well as CD150 KLS cells in the BM. Studies on characterization of the hematopoietic system of Postn-/- mice are underway. To further determine the role of ITGAV in HSCs, we used blocking antibodies against ITGAV and performed homing and engraftment studies. No effect on either homing potential or engraftment of ST- and LT- engraftment was seen. However, the competitive repopulation of ITGAV- CD150 KLS cells was significantly lower that that of ITGAV+ CD150 KLS cells (isolated using non-blocking antibodies). Therefore, our studies confirm the importance of ITGAV expression on primitive HSCs as well as presents POSTN as an important cell cycle regulator in the hematopoietic system. Disclosures: No relevant conflicts of interest to declare.

Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4185-4193 ◽  
Author(s):  
Hanno Glimm ◽  
IL-Hoan Oh ◽  
Connie J. Eaves
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Cell Activity ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
A Cell

An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G1fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G0/G1and S/G2/M. Interestingly, the G0 cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle–associated change in in vivo stem cell homing, the cultured G0/G1 and S/G2/M CD34+ CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor β1 that increased the G0/G1 fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle–associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo–manipulated grafts.

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 > 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 CDK6 Antagonizes TP53-Induced Responses during Tumorigenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-15-SCI-15
Author(s):  
Veronika Sexl ◽  
Karoline Kollmann ◽  
Florian Bellutti
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Conflicts Of Interest ◽  
Induced Responses ◽  
Hematopoietic Stem ◽  
Hematopoietic Malignancies ◽  
Independent Functions ◽  
Oncogenic Stress

Inhibitors directed against cyclin dependent kinases (CDKs) have raised much interest as anti-cancer therapeutics over the last years. In particular, inhibitors directed against CDK4/6 have been declared as a major breakthrough in cancer therapy by the FDA. CDK4 and CDK6 bind to D-type cyclins and subsequently phosphorylate the RB protein to allow cells to progress through the G1 phase of the cell cycle. The effectiveness of CDK4/6 inhibitors was primarily assigned to their ability to block cell cycle progression. In hematopoietic malignancies high levels of CDK6, but not CDK4, are frequently found. Over the last years we have assigned a novel and unexpected role for CDK6 as global transcriptional regulator. ChIP-Seq experiments identified more than 20.000 specific CDK6 binding sites in leukemic cells with the majority located in the promoter regions. CDK6 binding to chromatin does not require kinase activity whereas transcriptional control is regulated in a kinase- dependent as well as kinase-independent manner. Overlaying ChIP-Seq and RNA-Seq experiments showed that CDK6 contributes to the induction or repression of genes. Target genes of CDK6 which are important for leukemia progression include PIM1, c-MYC, AURKA, AURKB, AKT and VEGF-A. Murine leukemia models verified the importance of CDK6 for myeloid and lymphoid tumor formation downstream of a variety of oncogenes including FLT3-ITD, NPM/ALK, MLL/AF9, BCR/ABL or JAK2V617F. CDK6 contributes to disease development by regulating proliferation, cell survival, angiogenesis and cytokine production. In hematopoietic stem cells and leukemic stem cells kinase-independent functions dominate and CDK6 controls a network of transcription factors regulating stem cell quiescence and activation. The importance of kinase-dependent transcriptional effects is pronounced under conditions of stress and transformation. Upon oncogenic stress, CDK6 induces a set of genes that counteract pro-apoptotic TP53 responses including MDM4, PRMT5, PPM1D and BCL2. This response is induced by a CDK6 - dependent phosphorylation of the transcription factors SP1 and NFYA as verified by phospho-chromatome analysis. Murine Cdk6-deficient cells only survive oncogenic stress by mutating Tp53. The link between CDK6 and TP53 is conserved in human hematopoietic malignancies. Kollmann K, Heller G, Schneckenleithner C, et al. A kinase-independent function of CDK6 links the cell cycle to tumor angiogenesis. Cancer Cell. 2013;24(2):167-181.Scheicher R, Hoelbl-Kovacic A, Bellutti F, et al. CDK6 as a key regulator of hematopoietic and leukemic stem cell activation. Blood. 2015;125(1):90-101.Uras IZ, Walter GJ, Scheicher R, et al. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6. Blood. 2016;127(23):2890-2902.Bellutti F, Tigan AS, Nebenfuehr S, et al. CDK6 antagonizes P53-induced responses during tumorigenesis. Cancer Discov. 2018;8(7):884-897.Uras IZ, Maurer B, Nivarthi H, et al. CDK6 coordinates JAK2V617F mutant MPN via NF-kB and apoptotic networks. Blood. 2019;133(15):1677-1690. Disclosures No relevant conflicts of interest to declare.

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