Isolation and Functional Analysis of CXCR7 Promoter - a Novel Receptor for Stromal Derived Factor-1 (SDF-1): Different Regulation of Expression in Human Hematopoietic Cells Versus Pediatric Sarcomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4583-4583 ◽  
Author(s):  
Maciek Tarnowski ◽  
Magdalena Kucia ◽  
Mariusz Z Ratajczak
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Binding Site ◽  
Promoter Activity ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Pediatric Sarcomas

Abstract Abstract 4583 Stromal derived factor-1 (SDF-1) binds to seven transmembrane-span G-protein coupled receptor CXCR4 and directs homing of CXCR4+ hematopoietic stem cells to bone marrow (BM) as well as metastasis of CXCR4+ cancer cells. Recently, a new SDF-1 binding receptor has been identified and named CXCR7. With identification of this receptor a role of SDF-1 in directing chemotaxis of CXCR7+ hematopoietic cells as well as metastasis of CXCR7+ tumors become more complex. While CXCR7 is expressed at very low level on normal hematopoietic stem cells, its expression becomes high on leukemic cells. Similarly we noticed high expression of CXCR7 on several pediatric sarcomas (e.g., rhabdomyosarcomas and neuroblastomas) that very often metastasize/infiltrate BM. The aim of our study was to evaluate 5′ fragment of CXCR7 gene for promoter activity and analyze how its expression is regulated in CXCR7+ human hematopoietic cells as well as in CXCR7+ human rhabdomyosarcoma cells (RMS). The putative CXCR7 promoter was cloned by employing specific primers for 5′ fragment of CXCR7 gene. We found that this 2.5 kb 5′ DNA fragment adjacent to CXCR7 gene contains three potential hypoxia responsive element (HRE)- (-100-104, -965-969, -1306-1310), five NF-kB- (-32-42, -308-318, -1019-1029, -1375-1379, -2145-2155), four NRF-1 binding sites (-1030-1040, -1468-1478, -1980-1990, -2085-2095), one c-myb binding site at -15-19 and at -702-706 a binding site for negative transcription regulatory factor YY1. We generated 8 constructs containing smaller CXCR7 promoter fragments and three constructs containing mutated distal NF-kB and HREs as well as c-myb that were subcloned into a pGL4.10 vector. The promoter activity of these fragments was tested in transfected human hematopoietic cells (THP-1) and RMS cell line (RD) by measuring luciferase activity. While the minimal promoter activity in human hematopoietic cells was retained in 80 bp short fragment containing c-myb binding site, similar activity in human rhabdomyosarcoma cells required longer 150 bp fragment containing proximal NF-kB binding element. We noticed that while mutation of c-myb binding site in CXCR7 promoter in THP-1 cells reduces promoter activity by ∼50%, mutation of proximal NF-kB-binding site in CXCR7 promoter completely inhibits promoter activity in RD cells. This was confirmed by knock-down of c-myb by shRNA and chemical inhibition of NF-kB respectively. Furthermore, we noticed that during hypoxia in contrast to CXCR4, CXCR7 expression does not change in hematopoietic cells, however is significantly downregulated in rhabdomyosarcoma cells. This could be explained by upregulation of negative transcripton factor YY1 during hypoxia, as evidenced by RQ-PCR and confirmed by CHIP assay. In conclusion we have demonstrated that 5′ fragment of CXCR7 possesses promoter activity and is differently regulated in hematopoietic versus sarcoma cells - in c-myb or NF-kB dependent manner respectively. Furthermore, we also found that in contrast to hematopoietic cells hypoxia inhibits CXCR7 promoter activity in RMS cells in YY1-dependent manner. 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.

Competition In Engraftment of Normal Hematopoietic Stem Cells and Leukemic Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4836-4836
Author(s):  
Gyeongsin Park ◽  
Michael Heuser ◽  
Tobias Berg ◽  
R. Keith Humphries
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Fixed Number ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Abstract 4836 Engraftment is a process including homing to bone marrow, implantation and proliferation. Implantation implies interactions with specialized microenvironments, niches, in which hematopoietic stem cells (HSCs) live and are regulated. Studies have demonstrated the possibility that leukemic stem cells (LSCs) interact with niches in a similar manner to HSCs. We investigated whether HSCs and LSCs compete with each other in their engraftment. We employed a mouse transplantation assay with unmanipulatated bone marrow cells (BMCs) as a source of normal HSCs and LSCs generated by transduction of BMCs with Meningioma 1 (MN1), a potent oncogene causing myeloid leukemia in mice. In irradiated recipients (750 cGy), cotransplantation of leukemic cells (1×105) with various numbers of BMCs (1×105, 1×106 and 1×107) demonstrated that the engraftment level of leukemic cells is influenced by BMCs in a dose dependant manner (5.2%, 41.3% and 82.2% at 2-weeks; 52.3%, 69.5% and 86.9% at 4weeks; mice died before the 5 weeks bleeding, 94.9% and 97.5% at 5weeks, respectively). Cotransplantation of various numbers of leukemic cells (1×104, 1×105 and 1×106) with a fixed number of BMCs (1×106) demonstrated a similar pattern of leukemic engraftment (7.0%, 59.5% and 87.1% at 2weeks; 62.0%, 85.7% at 4 weeks, and mice died before the four week bleeding, respectively). To further elucidate the competition between HSCs and LSCs, we transplanted the cells at different time intervals. Transplantation of normal BMCs (1×106) 2 days prior to transplantation of LSCs (1×105) resulted in much reduced levels of leukemic engraftment compared to that seen in mice simultaneously transplanted (3.5% vs 59.5% at 2 weeks; 73.1% vs 85.76% at 4weeks). This competitive suppression of leukemic engraftment was further enhanced by transplanting larger numbers of normal BMCs (2×107) as little as 12 hours prior LSC transplantation (5×105) compared to simultaneous injection (0% vs 7.26% at 2weeks, 0.9% vs 35.3% at 3 weeks, and 6.0% vs 60.6% at 4 weeks). When BMCs (1×105) or leukemic cells (1×105) were transplanted at equal doses of 1×105 together with normal helper cells (1×106) the leukemic cells expanded 280-fold compared to only 7.3 fold for normal BMCs at 2 weeks (total cell count from two femurs and two tibias per 1×105 transplanted cells). Thus the competitive suppression of leukemic cell growth seen upon sequential transplantation of normal BMCs is not readily explained by enhanced kinetics of normal BMC growth but rather by competition at the level of initial engraftment. In conclusion, our data demonstrate that there is a competition between normal and leukemic cells during the engraftment process, suggesting niche competition of HSCs and LSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Aerobic exercise in humans mobilizes HSCs in an intensity-dependent manner

2017 ◽  
Vol 122 (1) ◽  
pp. 182-190 ◽  
Author(s):  
Jeff M. Baker ◽  
Joshua P. Nederveen ◽  
Gianni Parise
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mononuclear Cells ◽  
Gradient Centrifugation ◽  
Hematopoietic Cells ◽  
Exercise Group ◽  
Cell System ◽  
Dependent Manner ◽  
Total Blood ◽  
Hematopoietic Stem

Hematopoietic stem and progenitor cells are necessary to maintain, repair, and reconstitute the hematopoietic blood cell system. Mobilization of these cells from bone marrow to blood can be greatly increased under certain conditions, one such being exercise. The purpose of this study was to identify the importance of exercise intensity in hematopoietic mobilization, to better understand the mobilization kinetics postexercise, and to determine if exercise is capable of mobilizing several specific populations of hematopoietic cells that have clinical relevance in a transplant setting. Healthy individuals were exercised on a cycle ergometer at 70% of their peak work rate (WRpeak) until volitional fatigue and at 30% of their WRpeakwork matched to the 70% WRpeakbout. Blood was collected before, immediately post, and 10, 30, and 60 min postexercise. Total blood cells, hematocrit, and mononuclear cells isolated by density gradient centrifugation were counted. Specific populations of hematopoietic stem cells were analyzed by flow cytometry. Mononuclear cells, CD34+, CD34+/CD38−, CD34+/CD110+, CD3−/CD16+/CD56+, CD11c+/CD123−, and CD11c−/CD123+cells per millilter of blood increased postexercise. Overall, the 70% WRpeakexercise group showed greater mobilization immediately postexercise, while there was no observable increase in mobilization in the work matched 30% WRpeakexercise group. Mobilization of specific populations of hematopoietic cells mirrored changes in the general mobilization of mononuclear cells, suggesting that exercise serves as a nonspecific mobilization stimulus. Evidently, higher intensity exercise is capable of mobilizing hematopoietic cells to a large extent and immediately postexercise is an ideal time point for their collection.NEW & NOTEWORTHY Here we demonstrate for the first time that mobilization of hematopoietic stem cells (HSCs) through exercise is intensity dependent, with the greatest mobilization occurring immediately after high-intensity exercise. As well, we show that exercise is a general stimulus for mobilization: increases in specific HSC populations are reliant on general mononuclear cell mobilization. Finally, we demonstrate no differences in mobilization between groups with different aerobic fitness.

scholarly journals Maintenance of Hematopoietic Stem Cells Through Regulation of Wnt and mTOR Pathways.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2309-2309
Author(s):  
Jian Huang ◽  
Peter S. Klein
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
Glycogen Synthase ◽  
Dependent Manner ◽  
Mtor Inhibition ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 2309 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evidence for malignant transformation in acute myeloid leukemia at the level of early hematopoietic stem cells by cytogenetic analysis of CD34+ subpopulations

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 2906-2912 ◽  
Author(s):  
D Haase ◽  
M Feuring-Buske ◽  
S Konemann ◽  
C Fonatsch ◽  
C Troff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Malignant Transformation ◽  
Cell Sorting ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a heterogenous disease according to morphology, immunophenotype, and genetics. The retained capacity of differentiation is the basis for the phenotypic classification of the bulk population of leukemic blasts and the identification of distinct subpopulations. Within the hierarchy of hematopoietic development and differentiation it is still unknown at which stage the malignant transformation occurs. It was our aim to analyze the potential involvement of cells with the immunophenotype of pluripotent stem cells in the leukemic process by the use of cytogenetic and cell sorting techniques. Cytogenetic analyses of bone marrow aspirates were performed in 13 patients with AML (11 de novo and 2 secondary) and showed karyotype abnormalities in 10 cases [2q+, +4, 6p, t(6:9), 7, +8 in 1 patient each and inv(16) in 4 patients each]. Aliquots of the samples were fractionated by fluorescence-activated cell sorting of CD34+ cells. Two subpopulations, CD34+/CD38-(early hematopoietic stem cells) and CD34+/CD38+ (more mature progenitor cells), were screened for karyotype aberations as a marker for leukemic cells. Clonal abnormalities and evaluable metaphases were found in 8 highly purified CD34+/CD38-populations and in 9 of the CD34+/CD38-specimens, respectively. In the majority of cases (CD34+/CD38-, 6 of 8 informative samples; CD34+/CD38+, 5 of 9 informative samples), the highly purified CD34+ specimens also contained cytogenetically normal cells. Secondary, progression-associated chromosomal changes (+8, 12) were identified in the CD34+/CD38-cells of 2 patients. We conclude that clonal karyotypic abnormalities are frequently found in the stem cell-like (CD34+/CD38-) and more mature (CD34+/CD38+) populations of patients with AML, irrespective of the phenotype of the bulk population of leukemic blasts and of the primary or secondary character of the leukemia. Our data suggest that, in AML, malignant transformation as well as disease progression may occur at the level of CD34+/CD38-cells with multilineage potential.

scholarly journals Thrombopoietin Induces HOXA9 Nuclear Transport in Immature Hematopoietic Cells: Potential Mechanism by Which the Hormone Favorably Affects Hematopoietic Stem Cells

2004 ◽  
Vol 24 (15) ◽  
pp. 6751-6762 ◽  
Author(s):  
Keita Kirito ◽  
Norma Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Hematopoietic Stem Cells ◽  
Nuclear Translocation ◽  
Hematopoietic Cells ◽  
Mitogen Activated Protein Kinase ◽  
Mrna Levels ◽  
Hematopoietic Stem ◽  
Nuclear Localization Signals ◽  
Mitogen Activated Protein

ABSTRACT Members of the homeobox family of transcription factors are major regulators of hematopoiesis. Overexpression of either HOXB4 or HOXA9 in primitive marrow cells enhances the expansion of hematopoietic stem cells (HSCs). However, little is known of how expression or function of these proteins is regulated during hematopoiesis under physiological conditions. In our previous studies we demonstrated that thrombopoietin (TPO) enhances levels of HOXB4 mRNA in primitive hematopoietic cells (K. Kirito, N. Fox, and K. Kaushansky, Blood 102:3172-3178, 2003). To extend our studies, we investigated the effects of TPO on HOXA9 in this same cell population. Although overall levels of the transcription factor were not affected, we found that TPO induced the nuclear import of HOXA9 both in UT-7/TPO cells and in primitive Sca-1+/c-kit+/Gr-1− hematopoietic cells in a mitogen-activated protein kinase-dependent fashion. TPO also controlled MEIS1 expression at mRNA levels, at least in part due to phosphatidylinositol 3-kinase activation. Collectively, TPO modulates the function of HOXA9 by leading to its nuclear translocation, likely mediated by effects on its partner protein MEIS1, and potentially due to two newly identified nuclear localization signals. Our data suggest that TPO controls HSC development through the regulation of multiple members of the Hox family of transcription factors through multiple mechanisms.

scholarly journals Blood and Cancer: Cancer Stem Cells as Origin of Hematopoietic Cells in Solid Tumor Microenvironments

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1293 ◽  
Author(s):  
Ghmkin Hassan ◽  
Masaharu Seno
Keyword(s):  
Stem Cells ◽  
Tumor Microenvironment ◽  
Cancer Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cancer Cells ◽  
Solid Tumor ◽  
Immune Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Tumor Microenvironments

The concepts of hematopoiesis and the generation of blood and immune cells from hematopoietic stem cells are some steady concepts in the field of hematology. However, the knowledge of hematopoietic cells arising from solid tumor cancer stem cells is novel. In the solid tumor microenvironment, hematopoietic cells play pivotal roles in tumor growth and progression. Recent studies have reported that solid tumor cancer cells or cancer stem cells could differentiate into hematopoietic cells. Here, we discuss efforts and research that focused on the presence of hematopoietic cells in tumor microenvironments. We also discuss hematopoiesis from solid tumor cancer stem cells and clarify the notion of differentiation of solid tumor cancer stem cells into non-cancer hematopoietic stem cells.

scholarly journals Negative Regulation of the Differentiation of Flk2− CD34− LSK Hematopoietic Stem Cells by EKLF/KLF1

2020 ◽  
Vol 21 (22) ◽  
pp. 8448
Author(s):  
Chun-Hao Hung ◽  
Keh-Yang Wang ◽  
Yae-Huei Liou ◽  
Jing-Ping Wang ◽  
Anna Yu-Szu Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Knockout ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Interesting Correlation ◽  
Regulatory Effects ◽  
High Level

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2− CD34− Lin− Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor β subunit (CSF2RB). As a result, Flk2− CD34− LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2− CD34− LSK-HSCs.

Exhaustion of Donor Hematopoietic Stem Cells in Lethally-Irradiated Hosts Can Be Sbstantially Mitigated by Targeting p18INK4C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1200-1200
Author(s):  
Hui Yu ◽  
Youzhong Yuan ◽  
Xianmin Song ◽  
Feng Xu ◽  
Hongmei Shen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Total Period ◽  
Over Expression

Abstract Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.

NF-kB Family Proteins Participate in Multiple Steps of Hematopoiesis through Elimination of Reactive Oxygen Species (ROS).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1694-1694
Author(s):  
Soichi Nakata ◽  
Itaru Matsumura ◽  
Hirokazu Tanaka ◽  
Yusuke Satoh ◽  
Takumi Era ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Growth And Survival ◽  
Ros Accumulation ◽  
Dependent Growth ◽  
Induced Apoptosis

Abstract NF-kB family proteins have been reported to induce the expression of over 150 target genes, thereby crucially regulating immune responses, stress responses, and inflammation. These proteins also play important roles in cell growth and survival in various cell types. However, the precise roles of NF-kB in hematopoiesis and their mechanisms remain undetermined. To examine the roles for NF-kB family proteins in the growth and survival of hematopoietic cells, we expressed dominant negative NF-kB (IkBSR) in a murine IL-3-dependent cell line Ba/F3 using a Lac-inducible system, in which IkBSR was inducibly expressed by the IPTG treatment; this clone was designated Ba/F3/IkBSR. Furthermore, we introduced EPO receptor (R), TPOR, and G-CSFR/gp130 consisting of the extracelluar domain of G-CSFR and cytoplasmic domain of gp130 into this clone. At first, we confirmed that these clones could survive and proliferate under the cultures with IL-3, EPO, TPO, G-CSF, respectively. Although IPTG-induced IkBSR slightly suppressed IL-3- and EPO-dependent growth at low concentrations, it did not affect TPO- or gp130L-dependent growth, suggesting that NF-kB might not be so important for cytokine-dependent growth of hematopoietic cells. In contrast, IkBSR prominently enhanced factor-deprived apoptosis, which was accompanied by the ROS accumulation. To access the roles of ROS in IkBSR-enhanced apoptosis, we overexpressed ROS scavenger enzymes MnSOD and thioredoxin X (TRX) in Ba/F3/IkBSR, respectively. As a result, MnSOD and TRX significantly canceled IkB-SR-enhanced apoptosis, suggesting that ROS would be responsible for this apoptosis. We next analyzed the effects of IkBSR on the growth and survival of normal hematopoietic cells. When IkBSR was introduced into murine Lin−Sca-1+ hematopoietic stem/progenitor cells with the retrovirus system, it induced apoptosis even in the presence of appropriate cytokines. This apoptosis was also accompanied by the ROS accumulation due to the downregulated expression of anti-oxidants such as glutathione, MnSOD, glutathione peroxidase, and TRX. In addition, the expression of antiapoptotic BCl-2 family members, Bcl-XL, Bcl-2, and A1 was found to be repressed by IkBSR. However, since antioxidants such as MCI (3-methyl-1-phenyl-2-pyrazolin-5-one), N-acetylecysteine and TRX cancelled this apoptosis, ROS were supposed to be more important for IkBSR-induced apoptosis in normal hematopoietic stem/progenitor cells. To further analyze the roles for NF-kB proteins in the development of hematopoietic cells, we expressed IkBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from ES cells. When IkBSR was expressed at the stage of hemangioblasts, IkBSR induced apoptosis and inhibited the development of hematopoietic stem cells, which was also cancelled by MCI. Furthermore, when IkBSR was expressed after the development of hematopoietic stem cells, it also inhibited terminal differentiation towards granulocytes, erythrocytes, and megakaryocytes through ROS-mediated apoptosis; IkBSR inhibited granulopoiesis before the development of myeloblasts, erythropoiesis after the development of proerythroblasts, and megakaryopoiesis during polyploidization of megakaryocytes. These results indicate that NF-kB family proteins play essential roles to prevent apoptosis at multiple steps of hematopoiesis by eliminating ROS.

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