The F-Box Protein NIPA Limits Hematopoietic Stem Cell Survival and Transplantation Efficiency

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1175-1175
Author(s):  
Stefanie Kreutmair ◽  
Anna Lena Illert ◽  
Rouzanna Istvanffy ◽  
Christina Eckl ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Spleen Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Mitotic Entry ◽  
Stem Cell Maintenance ◽  
F Box Protein

Abstract Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase and contributes to the timing of mitotic entry. To examine the function of NIPA in vivo, we generated Nipa deficient animals, which are viable but sterile due to a defect in testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in Nipa deficient animals. FACS analyses of spleen cells and bone marrow (BM) showed differences in Leucocyte subpopulations. Measuring the CD4 and CD8 positivity within all Thy1.2+ cells, the balance in NIPA-/- T-lymphocytes is destabilised in favour of CD4 positive cells. Besides CD43/CD19 positive as well as CD43/B220 positive cells within all leukocytes are increased in NIPA deficient spleen cells. Analysing more primitive cells, FACS data of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA-/- mice (age > 20 month), where LSKs were reduced to 40% of wildtype (wt) littermates (p=0,0171). Additionally, in such older NIPA-/- mice, only half the number of multipotent myeloid progenitors were detected in comparison to wt mice. To examine efficient response of stem cells to myeloid depression, mice were treated with 5-FU four days before BM harvest. We found that in NIPA-/- mice, both the number of myeloid progenitors as well as the number of LSKs were severely reduced compared to those in wt levels after 5-FU treatment (p<0.001). Interestingly, the reduction of progenitors and LSK cells was not dependent on age of the NIPA ko mice, suggesting a role for NIPA in stem cell activation or regeneration. This statement was studied in vitro by methylcellulose assays with 10 000 BM cells seeded in methylcellulose with cytokines and replated for three times after 10 days. Nipa deficient hematopoietic progenitors showed a reduced ability to proliferate and differentiate into colonies compared to their controls with an increasing difference after each replating (p(third replating) < 0.0001). Dynamic cell cycle analysis of seeded BM cells with BRDU and PI uncovered delayed cell cycle progress and mitotic entry in NIPA-/- BM cells in contrast to wt BM cells. Using competitive BM transplantation assay we investigated the role of NIPA for hematopoietic reconstitution in vivo. These experiments showed that NIPA-/- BM cells were severely deficient in hematopoietic recovery as recipient mice of NIPA-/- BM cells showed only half the amount of donor-derived peripheral blood cells in contrast to recipient mice of wt BM cells after 4, 11, 17 and over 23 weeks after transplantation. Furthermore NIPA-/- cells contributed only 7% in BM of transplanted mice 6 month after transplantation compared to 33% in recipients transplanted with wt BM cells (p<0.005). To further explore this defect in hematopoietic repopulation capacity and apply to more primitive progenitors serial transplantation assays were conducted with LSK cells transplanted together with support BM cells. Taken together our results demonstrate a critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.

ETO2 Controls Hematopoietic Stem Cell Expansion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 396-396
Author(s):  
Stephane Barakat ◽  
Julie Lambert ◽  
Guy Sauvageau ◽  
Trang Hoang
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Short Term ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 396 Hematopoietic stem cells that provide short term reconstitution (ST-HSCs) as well as hematopoietic progenitors expand from a small population of long term hematopoietic stem cells (LT-HSCs) that are mostly dormant cells. The mechanisms underlying this expansion remain to be clarified. SCL (stem cell leukemia), is a bHLH transcription factor that controls HSC quiescence and long term competence. Using a proteomics approach to identify components of the SCL complex in erythroid cells, we and others recently showed that the ETO2 co-repressor limits the activity of the SCL complex via direct interaction with the E2A transcription factor. ETO2/CBF2T3 is highly homologous to ETO/CBFA2T1 and both are translocation partners for AML1. We took several approaches to identify ETO2 function in HSCs. We initially found by Q-PCR that ETO2 is highly expressed in populations of cells enriched in short-term HSC (CD34+Flt3-Kit+Sca+Lin-) and lympho-myeloid progenitors (CD34+Flt3+Kit+Sca+Lin-) and at lower levels in LT-HSCs (CD34-Kit+Sca+Lin- or CD150+CD48-Kit+Sca+Lin-). Next, the role of ETO2 was studied by overexpression or downregulation combined with transplantation in mice. Ectopic ETO2 expression induces a 100 fold expansion of LT-HSCs in vivo in transplanted mice associated with differentiation blockade in all lineages, suggesting that ETO2 overexpression overcomes the mechanisms that limit HSC expansion in vivo. We are currently testing the role of the NHR1 domain of ETO2 in this expansion. Conversely, shRNAs directed against ETO2 knock down ET02 levels in Kit+Sca+Lin- cells, causing a ten-fold decrease in this population after transplantation, associated with reduced short-term reconstitution in mice. Finally, proliferation assays using Hoechst and CFSE indicate that ETO2 downregulation affects cell division (CFSE) and leads to an accumulation of Kit+Sca+Lin-cells in G0/G1 state (Hoescht). In conclusion, we show that ETO2 is highly expressed in ST-HSCs and lymphoid progenitors, and controls their expansion by regulating cell cycle entry at the G1-S checkpoint. In addition, ETO2 overexpression converts the self-renewal of maintenance into self-renewal of expansion in LT-HSCs. Disclosures: No relevant conflicts of interest to declare.

Nucleotide Receptor P2Y14 Modulates Hematopoietic Stem Cell Response to Tissue Injury Altering Stem Cell Preservation and Tissue Recovery.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 679-679 ◽  
Author(s):  
Yoriko Saito ◽  
Eyal Attar ◽  
Samyukta Jana ◽  
David Dombkowski ◽  
Viktor Janzen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Chemical Injury ◽  
Cell Cycle Entry ◽  
Induced Apoptosis ◽  
Lethal Irradiation

Abstract P2 receptors are functionally diverse cell surface receptors that bind nucleotides adenine (ADP, ATP) and uridine (UDP, UTP). P2Y receptors are metabotropic G protein-coupled receptors that mediate vascular and immune responses to injury. We previously reported the differential expression cloning of the UTP-glycoconjugate receptor, P2Y14 from quiescent primary human bone marrow (BM) hematopoietic stem cells (HSCs). Using P2Y14−/− mice, we now report that the presence of P2Y14 protects HSCs from apoptosis in the face of cytotoxic chemical injury. P2Y14 null mice develop normally and showed no significant differences in peripheral blood cell counts, BM cellularity or the absolute number/proportion of lin−cKit+Sca1+ (LKS+) and CD34−/lowLKS+ (34-LKS+) cells compared to their wildtype littermates. Similarly, cell cycle status, in vitro colony-forming cell (CFC) capacity, in vivo homing and in vivo colony-forming unit-spleen (CFU-S) function were unaffected. Since the role of nucleotide receptors in injury response have been reported, we examined BM HSC content following IP injection of 200mg/kg cyclophosphamide (CTX) and found that the relative protection of LKS+ and 34-LKS+ cells from CTX-induced apoptosis was lost in P2Y14 null animals (WT LKS+: 12.7% AnnexinV+7AAD-, KO LKS+ 36.8% AnnexinV+7AAD−, n=5 each, p=0.004; WT 34-LKS+: 13.2% AnnexinV+7AAD−, KO LKS+ 38.7% AnnexinV+7AAD−, n=5 each, p=0.007). In addition, the kinetics of long-term myeloid recovery after a single injection of 5-Fluorouracil (5FU) IP 150mg/kg was significantly more accentuated in P2Y14 null animals, with significantly greater peripheral blood Gr-1+ cell count at days 21–56 post-injection (n=10 each, p=0.009). When sorted BM LKS+ cells were exposed in vitro to UDP-glucose, a putative P2Y14 ligand known to be released from cytoplasm during cellular injury, BrDU incorporation was significantly reduced (n=3 each, p&lt;0.05), suggesting that P2Y14 activation with UDP-glucose reduces HSC cell cycle entry in response to injury. While these in vivo models examine HSC response to injury to both BM microenvironment and the HSCs themselves, when uninjured HSCs were reintroduced into injured microenvironment in the setting of hematopoietic reconstitution following lethal irradiation, P2Y14 null BM HSCs performed better in serial transplantation (n=10 each, p&lt;0.01 for primary, secondary and tertiary transplantation), showing greater reconstitution and self-renewal capacity compared with WT littermates. From these findings, we propose that P2Y14 protects HSCs from chemical injury by acting as a sensor for metabolic “danger signal” in the form of released intracellular UDP-glucose during acute chemical injury in the BM and maintaining relative resistance of HSCs to toxin-induced apoptosis by restricting cell cycle entry. In the setting of injury exclusive to BM microenvironment (HSC transplantation), P2Y14 null HSCs, unable to detect UDP-glucose, respond to highly proliferative environment following lethal irradiation, resulting in greater reconstitution and self-renewal.

scholarly journals The APC/C Coactivator Cdh1 Controls Self-Renewal and Differentiation of Human and Murine HSPCs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2650-2650
Author(s):  
Daniel Ewerth ◽  
Stefanie Kreutmair ◽  
Andrea Schmidts ◽  
Marie Follo ◽  
Dagmar Wider ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Research Funding ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract Introduction: The balance between differentiation and self-renewal in hematopoietic stem and progenitor cells (HSPCs) is crucial for homeostasis and lifelong blood cell production. Differentiation is predominantly initiated in the G1 phase of the cell cycle when the E3 ligase anaphase-promoting complex or cyclosome (APC/C) is highly active. Its coactivator Cdh1 determines substrate specificity and mediates proteasomal degradation. Relevant target proteins are associated with cell fate decisions in G1/G0, and there is growing evidence that Cdh1 is an important regulator of differentiation. While this has already been demonstrated in neurons, muscle cells or osteoblasts, little is known about the role of APC/CCdh1 in hematopoiesis. Here we report on the function of Cdh1 in human and murine HSPCs in vitro and in vivo. Methods: Human CD34+ cells from the peripheral blood of G-CSF mobilized donors were exposed to different cytokine combinations and gains or losses of surface marker expression during cell division were determined. By using the established culture conditions Cdh1 expression was detected in distinct hematopoietic lineages and developmental states. CD34+ cells were transduced with a lentivirus to deplete Cdh1 by stably expressing shRNA and was then used for in vitro differentiation in liquid culture or CFU assay. In a second miR-based RNAi approach murine BM cells were depleted of Cdh1 and used for competitive transplantation assays. Complementary xenotransplantation of human Cdh1-depleted CD34+cells was carried out with NSG mice. Results: The stimulation of freshly thawed CD34+ cells with cytokines led to cell cycle entry and proliferation. Self-renewing cells preserved CD34 expression for up to 7 cell divisions with a low proliferation rate. In contrast, during granulopoiesis and erythropoiesis cells divided more frequently with rapid down-regulation of CD34. Cdh1 expression was tightly connected to differentiation status and proliferation properties. In vitro cultured CD34+ cellsand those from BM of healthy human donors showed the highest Cdh1 level compared to moderate or low expression in lymphoid and myeloid cells. Cdh1 is highly expressed at the transcriptional and translational level during both self-renewal and also when cells were directed toward erythroid differentiation. Therefore, high Cdh1 expression is characteristic of immature hematopoietic cells and differentiating precursors. The knockdown of Cdh1 (Cdh1-kd) did not affect proliferation or viability as detected by CFSE staining and measuring the cell cycle length via live-cell imaging. However, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with a lower frequency of glycophorin A+ cells. The functional relevance of Cdh1 depletion was verified in CFU assays. Cells with Cdh1-kd formed fewer primary colonies but significantly more secondary colonies, indicating a preference for self-renewal over differentiation. After competitive transplantation Cdh1-depleted murine BM cells showed a significant enhancement in the repopulation of PB, BM and spleen at week 3, while there was no change in cell cycle properties. However, after 8 weeks chimerism in each of the compartments was reduced to that of the control cells. Accordingly, higher LK and LSK frequencies supported the engraftment of Cdh1-depleted cells at week 3, but there was a significant decrease at week 8 compared to control cells, suggestive of stem cell exhaustion. The Cdh1 level also affected cell differentiation in vivo. After 8 weeks the population of B cells (B220+) was increased in transplanted Cdh1-kd cells and the frequency of mature granulocytes (CD11b+ Gr1high) was reduced. Consistently, human Cdh1-depleted CD34+ cells engrafted to a much higher degree in the murine BM 8 and 12 weeks after xenotransplantation, as shown by a higher frequency of human CD45+ cells. Moreover, the increase of human CD19+ B cells with Cdh1-kd confirmed the results of the competitive transplantation. Conclusions: Loss of the APC/C coactivator Cdh1 supports repopulation of murine HSPCs after transplantation with a lymphoid-biased differentiation, and was confirmed in xenotranplantation experiments. In the long-term, Cdh1 loss led to exhaustion of primitive LK and LSK population, highlighting the role of Cdh1 as a critical regulator of HSPC self-renewal and differentiation. Disclosures Engelhardt: Janssen: Research Funding; Amgen: Research Funding; MSD: Research Funding; Celgene: Research Funding.

Enforced Nr4a2 expression Drives HSCs into Quiescence.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1326-1326
Author(s):  
Olga Sirin ◽  
Orla M Conneely ◽  
Margaret A. Goodell
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Biology ◽  
Bone Marrow Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Proliferation Capacity

Abstract Hematopoietic stem cells (HSCs) are used as a paradigm for understanding somatic stem cell biology, yet the factors that regulate their self-renewal and differentiation are poorly understood. Our lab has recently discovered that within the hematopoietic lineage, Nr4a2 expression is restricted to the stem and progenitor cell compartment. Furthermore, we have observed a dramatic decrease in its expression when the bone marrow cells are exposed to 5-fluorouracil which forces quiescent HSCs into cycle. We therefore hypothesize that Nr4a2 may be involved in maintaining stem cell quiescence. To better understand the role of Nr4a2 in hematopoiesis, we have performed gain-of-function studies in which Nr4a2 is overexpressed in HSCs. Bone marrow transplantation (BMT) experiments revealed that Nr4a2 overexpressing cells contributed significantly less to the peripheral blood compared to the control (1% vs 35%, see fig). This suggests that enforced Nr4a2 expression negatively affects HSC regeneration potential. We also found that Nr4a2 overexpression has a negative effect on the overall proliferation capacity with an in vitro methocult assay. Together, these results support our hypothesis that the high level expression of Nr4a2 regulates HSC quiescence by inhibiting HSC self-renewal and/or differentiation. Cell cycle analysis demonstrates that enforced expression of Nr4a2 causes an increase in the number of cells in quiescence. These data are supported further by analysis at a molecular level revealing that overexpression of Nr4a2 increases expression of cell cycle inhibitors such as p18 and p19, both of which are important for Go to G1 entry. By uncovering the role of Nr4a2 in hematopoiesis we aim to achieve not only a better understanding of the regulation of stem cell self-renewal and differentiation but also of the pathways involved in HSC cell cycle regulation. Figure Figure

Evi1 Is a Stem Cell-Specific Regulator of Self-Renewal Capacity In the Definitive Hematopoietic System

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 838-838
Author(s):  
Keisuke Kataoka ◽  
Tomohiko Sato ◽  
Akihide Yoshimi ◽  
Susumu Goyama ◽  
Takako Tsuruta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Specific Loss ◽  
Loss Of Self ◽  
Colony Forming Capacity

Abstract Abstract 838 Self-renewal is a defining property of stem cells. Although a number of molecules have been implicated in the regulation of hematopoietic stem cell (HSC) self-renewal, loss of these genes is accompanied with other hematological abnormalities. Thus, it is unclear what will happen with a specific loss of self-renewal capacity of HSCs. Evi1 is an oncogenic transcription factor in myeloid malignancies. Evi1 expression is limited to hematopoietic stem/progenitor fraction, and Evi1 is essential for the maintenance of HSCs, but is dispensable for blood cell lineage commitment. Thus, we hypothesized that Evi1 expression could distinguish hematopoietic stem and progenitor cells, and reduction of Evi1 gene dosage might cause a specific loss of self-renewal activity. First, to elucidate Evi1 expression within the hematopoietic system, we have generated Evi1-IRES-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of Evi1 gene. We found that Evi1 was predominantly expressed in the hematopoietic stem/progenitor fraction (Lin- Sca-1+ c-kit+ (LSK)), but its expression was rapidly extinguished during early stages of lineage commitment. Among the LSK compartment, Evi1 was expressed at the highest level in long-term HSCs (LT-HSCs; Flk2- CD34-, CD48- CD150+, or SP-tip fractions in LSK cells). Next, we hypothesized that Evi1 would have the potential to mark LT-HSCs effectively. To test this, we compared GFP+ and GFP- cells in the LSK fraction, and revealed that GFP+ LSK cells were more immature and quiescent with a higher colony-forming capacity than GFP- LSK cells. In addition, in vivo long-term multilineage repopulating cells were exclusively enriched in the GFP+ LSK fraction. In the embryo, Evi1 was highly expressed in the hematopoietic stem/progenitor fraction; that is, CD34+ c-kit+ cells in embryonic day 10.5 (E10.5) aorta-gonad-mesonephros, CD34+ c-kit+ CD48- cells in E12.5 placenta, and Mac-1+ Sca-1+ Lin- (MSL) CD48- cells in E14.5 fetal liver (FL). In vivo competitive repopulation assay showed that, in the MSL fraction of FL, GFP+ MSL cells exclusively had a long-term multilineage repopulating capacity. These results implied that Evi1 plays a more specific role in HSCs than in other hematopoietic cells. To clarify this, we analyzed heterozygous Evi1 knockout mice (Evi1 +/− mice), as it seems difficult to elucidate the function of a small population of HSCs in Evi1 conditional knockout mice due to the leaky expression of Cre recombinase. We have previously showed that haploinsufficiency of Evi1 leads to decreased numbers of LSK and CD34- LSK cells, and impaired long-term repopulating activity. Here we demonstrated the number of each fraction in Evi1 +/− LSK cells was reduced in proportion to their expression level of Evi1. But, there were no significant differences in the numbers of lymphoid and myeloid progenitors between Evi1 +/+ and Evi1 +/− mice. Evi1 +/− CD34+ LSK cells had an equivalent in vitro colony-forming capacity and day 11 colony-forming unit-spleen activity to Evi1 +/+ CD34+ LSK cells. However, in vivo short-term repopulation assay using CD34+ LSK cells showed that the percentage of donor-derived cells from Evi1 +/− mice was significantly declined at 4 weeks after transplantation. Moreover, Evi1 +/− CD34- LSK cells had a pronouncedly impaired in vivo repopulating capacity. These data suggested that the differentiation capacity of Evi1 +/− HSCs was maintained, but their self-renewal capacity was specifically reduced. Although flow cytometric analysis of cell-cycle status and apoptosis showed no differences in CD34- LSK cells between Evi1+/+ and Evi1 +/− mice, the G0 fraction of Evi1 +/− CD34+ LSK cells was significantly reduced, indicating that these cells might proliferate more rapidly to compensate for the impaired self-renewal capacity of HSCs. In conclusion, we showed that Evi1 is predominantly expressed in HSCs and its expression can mark long-term repopulating HSCs in the fetal and adult hematopoietic system. Moreover, functional loss caused by haploinsufficiency of Evi1 is limited to a defect of self-renewal capacity of HSCs, and the increased cell-cycle progression of CD34+ LSK cells in Evi1 +/− mice seems to be the consequence of the impaired self-renewal capacity. Our data may help to understand the unrevealed effects of loss of self-renewal activity of HSCs and compensative mechanism of their defects. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Act of Controlling Adult Stem Cell Dynamics: Insights from Animal Models

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 667
Author(s):  
Meera Krishnan ◽  
Sahil Kumar ◽  
Luis Johnson Kangale ◽  
Eric Ghigo ◽  
Prasad Abnave
Keyword(s):  
Stem Cells ◽  
Animal Models ◽  
Adult Stem Cells ◽  
The Body ◽  
In Vivo Studies ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Organ Systems

Adult stem cells (ASCs) are the undifferentiated cells that possess self-renewal and differentiation abilities. They are present in all major organ systems of the body and are uniquely reserved there during development for tissue maintenance during homeostasis, injury, and infection. They do so by promptly modulating the dynamics of proliferation, differentiation, survival, and migration. Any imbalance in these processes may result in regeneration failure or developing cancer. Hence, the dynamics of these various behaviors of ASCs need to always be precisely controlled. Several genetic and epigenetic factors have been demonstrated to be involved in tightly regulating the proliferation, differentiation, and self-renewal of ASCs. Understanding these mechanisms is of great importance, given the role of stem cells in regenerative medicine. Investigations on various animal models have played a significant part in enriching our knowledge and giving In Vivo in-sight into such ASCs regulatory mechanisms. In this review, we have discussed the recent In Vivo studies demonstrating the role of various genetic factors in regulating dynamics of different ASCs viz. intestinal stem cells (ISCs), neural stem cells (NSCs), hematopoietic stem cells (HSCs), and epidermal stem cells (Ep-SCs).

scholarly journals Replication Stress Response and CDKN1A Engagement Constrain Fetal Hematopoietic Stem Cell Pool Size in Fanconi Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 107-107
Author(s):  
Makiko Mochizuki-Kashio ◽  
Young Me Yoon ◽  
Theresa N Menna ◽  
Markus Grompe ◽  
Peter Kurre
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Nuclear Localization ◽  
Fanconi Anemia ◽  
Fetal Liver ◽  
Replication Stress ◽  
Pool Size ◽  
Hematopoietic Stem ◽  
Independent Functions

Bone marrow (BM) failure is the principal source of morbidity and mortality in Fanconi Anemia (FA) patients. Recessively inherited germline mutations in one of 25 genes lead to deficits by in a pathway central to DNA crosslink repair. Functionally, FA proteins protect adult hematopoietic stem cells (HSC) from p53 mediated apoptosis elicited by alkylating agents, a range of experimental inflammatory cues or aldehyde exposure. However, these mechanisms do not seem to account for depleted hematopoietic stem and progenitor cell pools in very young FA patients, or the spontaneous, non-apoptotic and p53-independent fetal HSC deficits observed in murine models. Building on our previous observation of a quantitatively constrained fetal HSC pool in FA mice (Fancd2-/-), the current experiments reveal the specific developmental timeframe for the onset of stem cell deficits during HSC expansion in the fetal liver (FL). Cell cycle studies using an EdU/BrdU pulse chase protocol reveal delays in S-phase entry and progression at E13.5. Building on the role of FA proteins (FANCM, FANCI and FANCD2) in countering experimental replication stress (RS) in cell line models, we reasoned that rapid rates of proliferation required during expansion in the FL may similarly confer RS on the FA HSC pool. Experiments in E13.5 FL HSC confirmed the predicted increase in single stranded DNA and accumulation of nuclear replication associated protein (pRpa), along with activation of pChk1, a critical cell cycle checkpoint in cells under RS. For comparison, pChk1 in unperturbed adult cells was no different between Fancd2-/- and WT. The data are also consistent with gains in RAD51 and alkaline comet assays we previously published (Yoon et al., Stem Cell Reports 2016). The cell cycle regulator Cdkn1a (p21) is considered a canonical downstream component of the p53 response in adult FA HSC, but it also performs p53 independent functions in the RS response that coincide with its role in the nucleus. Here, we observed an increase in nuclear localization of p21 in Fancd2-/- FL HSC. TGF-β is a critical developmental morphogen that regulates p21 activity, and TGF-β inhibitors can partially reverse adult FA HSC function along with suppression of NHEJ mediated DNA repair. To test regulation of p21 in fetal HSC under RS, we first treated WT FL HSC with aphidicolin to experimentally simulate RS and found that SD208, a small molecule TGF-β-R1 inhibitor, completely rescued the p21 nuclear localization. We then went on to demonstrate that pharmacological inhibition of TGF-β signaling also reversed the nuclear p21 translocation in FA FL HSC (under physiological RS) and functionally rescued the primitive myeloid progenitor colony formation (CFU-GEMM) in vitro. Altogether, our data show that HSC deficits in FA first emerge in the fetal liver, where rapid fetal expansion causes RS that elicits pChk1 activation and nuclear p21 translocation, which restrain cell cycle progression and act as principal mechanisms limiting fetal HSC pool size in FA. Our experiments suggest a central and p53-independent role for p21 in fetal FA HSC regulation. Detailed knowledge of the physiological role of FA proteins in fetal phenotype HSC has the potential to lead to new therapies that delay or rescue hematopoietic failure in FA patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1204-1204
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Nathanael G Bailey ◽  
Meiling Zhao ◽  
Kevin B Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Double Mutant ◽  
Mutant Mice ◽  
Cytokine Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Mutant ◽  
Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Adenosine from Niche-Associated Tregs Maintains Hematopoietic Stem Cell Quiescence

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 91-91
Author(s):  
Yuichi Hirata ◽  
Kazuhiro Furuhashi ◽  
Hiroshi Ishi ◽  
Hao-Wei Li ◽  
Sandra Pinho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pool Size ◽  
Immune Privilege ◽  
Haematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Conditional Deletion ◽  
Radiation Induced

Abstract A crucial player in immune regulation, FoxP3+ regulatory T cells (Tregs) are drawing attention for their heterogeneity and noncanonical functions. For example, specific subsets of Tregs in the adipose tissue control metabolic indices; muscle Tregs potentiate muscle repair, and lung Tregs prevent tissue damage. These studies, together with a previous finding that Tregs are enriched in the primary site for hematopoiesis, the bone marrow (BM), prompted us to examine whether there is a special Treg population which controls hematopoietic stem cells (HSCs). We showed that HSCs within the BM were frequently adjacent to distinctly activated FoxP3+ Tregs which highly expressed an HSC marker, CD150. Moreover, specific reduction of BM Tregs achieved by conditional deletion of CXCR4in Tregs, increased reactive oxygen species (ROSs) in HSCs. The reduction of BM Tregs further induced loss of HSC quiescence and increased HSC numbers in a manner inhibited by anti-oxidant treatment. Additionally, this increase in HSC numbers in mice lacking BM Tregs was reversed by transfer of CD150high BM Tregs but not of CD150low BM Tregs. These results indicate that CD150high niche-associated Tregs maintain HSC quiescence and pool size by preventing oxidative stress. We next sought to identify an effector molecule of niche Tregs which regulates HSCs. Among molecules highly expressed by niche Tregs, we focused on CD39 and CD73, cell surface ecto-enzymes which are required for generation of extracellular adenosine, because 1) CD39highCD73high cells within the BM were prevalent among CD150high Tregs and 2) HSCs highly expressed adenosine 2a receptors (A2AR). We showed that both conditional deletion of CD39 in Tregs and in vivo A2AR antagonist treatment induced loss of HSC quiescence and increased HSC pool size in a ROS-dependent manner, which is consistent with the findings in mice lacking BM Tregs. In addition, transfer of CD150high BM Tregs but not of CD150low BM Tregs reversed the increase in HSC numbers in FoxP3cre CD39flox mice. The data indicate that niche Treg-derived adenosine regulates HSCs. We further investigated the protective role of niche Tregs and adenosine in radiation injury against HSCs. Conditional deletion of CD39 in Tregs increased radiation-induced HSC apoptosis. Conversely, transfer of as few as 15,000 CD150high BM Tregs per B6 mouse (iv; day-1) rescued lethally-irradiated (9.5Gy) mice by preventing hematopoiesis failure. These observations indicate that niche Tregs protect HSCs from radiation stress. Finally, we investigated the role of niche Tregs in allogeneic (allo-) HSC transplantation. Our previous study showed that allo-hematopoietic stem and progenitor cells but not allo-Lin+ cells persisted in the BM of non-conditioned immune-competent recipients without immune suppression in a manner reversed by systemic Treg depletion1. This observation suggests that HSCs have a limited susceptibility to immune attack, as germline and embryonic stem cells are located within immune privileged sites. Because the study employed systemic Treg depletion and non-conditioned recipients, it remains unknown whether niche Tregs play a critical role in immune privilege of HSCs and in allo-HSC engraftment following conditioning. We showed here that the reduction of BM Tregs and conditional deletion of CD39 in Tregs abrogated allo-HSC persistence in non-conditioned immune-competent mice as well as allo-HSC engraftment following nonmyeloablative conditioning. Furthermore, transfer of CD150high BM Tregs but not of other Tregs (15,000 cells/recipient; day -2) significantly improved allo-HSC engraftment. This effect of niche Treg transfer is noteworthy given that 1-5 million Tregs per mouse were required in case of transfer of spleen or lymph node Tregs. These observations suggest that niche Tregs maintain immune privilege of HSCs and promote allo-HSC engraftment. In summary, our studies identify a unique niche-associated Treg subset and adenosine as regulators of HSC quiescence, numbers, stress response, engraftment, and immune privilege, further highlighting potential clinical utility of niche Treg transfer in radiation-induced hematopoiesis failure and in allo-HSC engraftment (under revision in Cell Stem Cell). 1 Fujisaki, J. et al. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature474, 216-219, doi:10.1038/nature10160 (2011). Disclosures No relevant conflicts of interest to declare.

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