Microbiota Signals Suppress the Lymphoid Specification of Hematopoietic Stem Cells

Abstract Aging is associated with an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced self-renewal capacity and myeloid-skewed lineage differentiation. Signals from commensal flora support basal myelopoiesis in young mice; however, their contribution to hematopoietic aging is largely unknown. Here, we characterize hematopoiesis in young and middle-aged mice housed under specific pathogen free (SPF) and germ-free (GF) conditions. We did not analyze older mice due to the difficulty in maintaining mice in a gnotobiotic facility for more than one year. Consistent with prior studies, there is a shift in hematopoiesis in aged SPF mice towards granulopoiesis, with a significant increase in the percentage of granulocytic cells and a decrease in B lineage cells in the bone marrow. The marked shift from lymphopoiesis to myelopoiesis that develops during aging of SPF mice is mostly abrogated in GF mice. Compared with aged SFP mice, there is a marked expansion of B lymphopoiesis in aged GF mice, which is evident at the earliest stages of B cell development. To investigate the impact of microbiota signals on multipotent HSPCs, we first quantified HSPCs by flow cytometry (Figure 1A-B). In aged SPF mice, the number of lineage - Sca1 + cKit + CD150 + CD48 - (LSK-SLAM) cells and CD34 - LSK-SLAM cells is increased 6.4 ± 1.7-fold and 3.4 ± 1.2-fold, respectively. Similar increases were observed in aged GF mice, with LSK-SLAM increasing 5.3 ± 1.6-fold (p=NS compared to SPF mice) and CD34 - LSK-SLAM cells increasing 2.8 ± 0.31-fold (p=NS). To quantify functional HSCs, limiting dilution transplantation experiments using unsorted bone marrow cells was performed. Although on a per cell basis the repopulating activity of aged HSCs is reduced, due to the large increase in phenotypic HSCs, the number of functional HSCs actually increases with aging, with similar increases in functional HSCs in aged SPF and GF mice (Figure 1C). Finally, to assess lineage-bias, we transplanted a limiting number of sorted HSCs and assessed lineage output. As expected, in young SPF mice, the majority of HSCs displayed a balanced myeloid/lymphoid lineage output, with a significant increase in myeloid-biased HSCs observed with aging (Figure 1D). In young GF mice, the majority of HSCs are lymphoid-biased. Moreover, although the myeloid output increased modestly with aging, the majority of HSCs in aged GF remained lymphoid-biased or balanced. Consistent with these data, RNA expression profiling of phenotypic HSCs from aged GF mice show enrichment for non-myeloid biased HSCs. Surprisingly, the RNA expression profiling data also suggest that inflammatory signaling is increased in aged GF HSCs compared with aged SPF HSCs. Collectively, these data suggest that microbiota-related signals suppress the lymphoid potential of HSCs, contributing to the expansion of myeloid-biased HSCs that occurs with aging. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Impact of Infusion Rate on the Early Engraftment Following Allogeneic Intra Bone Marrow Infusion of Hematopoietic Stem Cells in a Canine DLA-Identical Reduced Intensity Transplantation Model

Blood ◽

10.1182/blood.v126.23.5406.5406 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5406-5406

Author(s):

Stephanie Schaefer ◽

Juliane Werner ◽

Sandra Lange ◽

Katja Neumann ◽

Christoph Machka ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Infusion Rate ◽

Conflicts Of Interest ◽

Conditioning Regimen ◽

Hematopoietic Stem ◽

Time Points ◽

The Impact ◽

Reduced Intensity

Abstract Introduction: Direct intra bonemarrow (IBM) infusion of hematopoietic stem cells (HSC) is assumed to improve the homing efficiency and to accelerate the early engraftment in comparison to the conventional intravenous application of HSC. Especially for transplantation of low cell numbers i.e. "weak grafts" that is generally associated with delayed engraftment. The direct infusion of HSC in close proximity to the HSC niche by intra bone marrow transplantation (IBMT) might be a promising way. Whether the HSC infusion rate might influence the homing process and therefore the outcome after IBMT is so far unknown. Aims: Herein, we analyzed in a canine DLA-identical littermate model the impact of different graft infusion rates on the hematopoietic recovery as well as on the engraftment kinetics after IBMT following reduced intensity conditioning. Methods: Recipient dogs received IBMT following a 4.5 Gy total body irradiation (TBI). From day (d) -1 until d+35 Cyclosporin A (15mg/kg) was administered orally twice a day as immunosuppression. For IBM transfusion the graft volume was reduced by buffy coat centrifugation and dogs obtained 2x25 ml simultaneously into the humerus and femur. The infusion rate of the graft was 25ml/10 min in group 1 (IBM10, n = 8) and 25 ml/60 min in group 2 (IBM60, n = 7). A 28 day follow-up is currently available for twelve dogs (IBM10 n = 7; IBM60 n = 5). The development of the peripheral blood mononuclear cell (PBMC) and granulocyte chimerism was tested weekly. Blood count, kidney and liver enzymes were monitored routinely. Results: All animals engrafted. One dog of the IBM10 group died at d+15 (infection) and was therefore not included into analysis. The median number of infused total nucleated cells were in IBM10 4.1*108/kg (range 2.3-6.0*108/kg) and in IBM60 3.2*108/kg (range 1.8-4.4*108/kg; p=0.4). The infused CD34+ numbers were median 3.2*106/kg (range: 1.2-10.0*106/kg; IBM10) and 3.6*106/kg (range: 1.5-6.8*106/kg; IBM60; p=0.7). Time of leukocyte recovery was median d+11 after IBMT in both groups (range: d+4 to d+11, IBM10; d+8 to d+14, IBM60; p= 0.5). Median leukocytes nadirs amounted to 0.2*109/l for IBM10 and 0.3*109/l for IBM60 (p= 0.08). The median duration of leukopenia (<1*109/l) were similar (6d, range: 4-11d, IBM10; 3-9d, IBM60) (p= 0.6). Median platelet nadir was 0*109/l for both cohorts (range: 0.0-7.0*109/l, IBM10; 0.0-1.0*109/l, IBM60). The period of thrombocytopenia (≤20.0*109/l) was significantly prolonged in the IBM60 group (median 10d, range) compared to 5d (range: 3-12d) in the IBM10 group (p=0.05). Donor PBMC chimerisms at d+7, d+14 and d+28 were median 22% (range: 8-34%), 50% (range: 29-53%) and 67% (range: 47-73%) in IBM10. The results of PBMC chimerism for IBM60 were 11% (range: 5-34%), 42% (range: 20-42%) and 59% (range: 44-66%) at these time points (p = n.s.). Donor granulocyte chimerisms of median 33% (range: 11-83%), 100% (range: 58-100%) and 100% (range: 82-100%) were detected at d+7, d+14 and d+28 after HSCT in IBM10, respectively. The granulocyte chimerism in IBM60 amounted to 34% (range: 3-87%), 96% (range: 94-100%) and 98% (range: 96-100%) at the above mentioned time points p=n.s. for all time points). Conclusion: Our data suggest that early granulocyte and PBMC engraftment is not influenced by modification of the HSC infusion rate. However, the period of thrombocytopenia seems to be prolonged following a 60 minutes application. Therefore, longer infusion times in an IBMT setting seem not to be beneficial following toxicity reduced conditioning regimen. Disclosures No relevant conflicts of interest to declare.

Download Full-text

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

Blood ◽

10.1182/blood.v116.21.4836.4836 ◽

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.

Download Full-text

Ang1 Functions as An Autocrine Activating Factor of Tie2 Signaling In Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1548.1548 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1548-1548

Author(s):

Haruka Momose ◽

Kazuya Takizawa ◽

Madoka Kuramitsu ◽

Takuo Mizukami ◽

Atsuko Masumi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Molecular Mechanisms ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Down Regulation ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 1548 Hematopoietic stem cells (HSCs) are clonogenic cells that possess the self-renewal capacity to produce more HSCs, as well as the multilineage potential that gives rise to a defined set of mature differentiated progeny for maintenance or repair of the whole blood system. HSCs lie in the hematopoietic niches located along the inner surface of the bone or the sinusoidal endothelium, and are in contact with niche cells. The cell-cell interactions with niche cells are believed to be an important prerequisite to trigger signaling events in HSCs, thereby controlling the balance between HSC self-renewal and differentiation. However, the precise molecular mechanisms regulating niche cell-HSC interactions are not well understood. One of the key molecules for those interactions is Angiopoietin-1 (Ang1). Ang1 is expressed by the niche cells and has been identified as an activating ligand for Tie2 (tyrosine kinase with Ig-like loops and epidermal growth factor homology domains 2). The expression of Tie2 is dominant in HSCs, and Tie2 in HSCs is supposed to be stimulated by Ang1 derived from niche cells. However, Ang1 is also expressed in HSCs. Detailed analysis has shown that Ang1 expression was found to be restricted in long-term HSCs (CD34-lineage-Sca-1+c-Kit+), indicating that Ang1 derived from HSCs plays a role in regulating HSCs. We attempted to elucidate a novel regulating system for HSCs through Ang1-Tie2 signaling by utilizing a hematopoietic cell line in which Tie2 was stably expressed (Ba/F3-Tie2). In Ba/F3-Tie2 cells, Tie2 was found to be phosphorylated on tyrosine residues, even without exogenous addition of Ang1. In the same cells, the expression level of endogenous Ang1 was increased four-fold. When Ang1 expression was down-regulated by transduction with a lentiviral vector expressing short hairpin RNA (shRNA) for Ang1 (shAng1), the phosphorylation of Tie2 was suppressed, suggesting that Tie2 expressed in Ba/F3-Tie2 cells could be stimulated by endogenous Ang1. To mimic the physiological circumstances of the bone marrow, Ba/F3-Tie2 cells were cultured on OP9 stromal cells. Under these culture conditions, the effect of endogenous Ang1 was investigated. Down-regulation of Ang1 by shAng1 demonstrated an approximate 50% reduction in the proliferation of Ba/F3-Tie2 cells on the OP9 cell layer. A HSC-rich population of cells prepared from bone marrow (lineage-Sca-1+c-Kit+; LSK) was also analyzed on OP9 cell layers. Similar to the results obtained from the analysis of Ba/F3-Tie2 cells, down-regulation of Ang1 by shAng1 resulted in an approximately 70% decrease in the proliferation of LSK cells cultured on OP9 monolayers. We confirmed that the suppressive effect on HSC proliferation was due to the lack of Ang1 from HSCs by culturing on Ang1-defective OP9 cells. Finally, we performed in vivo analysis to confirm the importance of endogenous Ang1 to HSCs. Ly5.2 LSK cells transduced with the shAng1 expressing vector were transplanted along with Ly5.1xLy5.2 bone marrow cells into lethally irradiated Ly5.1 mice. The Ly5.2 donor-derived cells in the recipient's peripheral blood were monitored every 2 weeks. As expected, shAng1-introduced donor cells were at decreased ratios at week four (mean ratios, 31.5% for control vs. 17.5% for shAng1), and were reduced to an even lower level at week 12 (mean ratios, 27.1% for control vs. 6.79% for shAng1). This phenomenon was also confirmed by histochemical results, where statistically fewer HSCs existed in the bone marrow of recipient mice in which shAng1-introduced HSCs were transplanted, as compared to the control. Altogether, our data suggested that Tie2 in HSCs could be stimulated by the Ang1 produced by the surrounding HSCs, and this possible autocrine regulation might control the functions of HSCs. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Impact of Age and Gender on Hematopoietic Stem Cells and Immune Contexture of the Bone Marrow Microenvironment

Cells Tissues Organs ◽

10.1159/000510774 ◽

2020 ◽

pp. 1-6

Author(s):

Rebar N. Mohammed

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Immune Cells ◽

Absolute Number ◽

Cell Number ◽

Hematopoietic Stem ◽

The Absolute ◽

And Gender ◽

The Impact

Hematopoietic stem cells (HSCs) are a rare population of cells that reside mainly in the bone marrow and are capable of generating and fulfilling the entire hematopoietic system upon differentiation. Thirty-six healthy donors, attending the HSCT center to donate their bone marrow, were categorized according to their age into child (0–12 years), adolescence (13–18 years), and adult (19–59 years) groups, and gender into male and female groups. Then, the absolute number of HSCs and mature immune cells in their harvested bone marrow was investigated. Here, we report that the absolute cell number can vary considerably based on the age of the healthy donor, and the number of both HSCs and immune cells declines with advancing age. The gender of the donor (male or female) did not have any impact on the number of the HSCs and immune cells in the bone marrow. In conclusion, since the number of HSCs plays a pivotal role in the clinical outcome of allogeneic HSC transplantations, identifying a younger donor regardless the gender is critical.

Download Full-text

Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽

10.1182/blood.v84.1.74.74 ◽

1994 ◽

Vol 84 (1) ◽

pp. 74-83 ◽

Cited By ~ 36

Author(s):

SJ Szilvassy ◽

S Cory

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Bone Marrow Cells ◽

High Titer ◽

Marker Gene ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

Download Full-text

Transplantation of hematopoietic stem cells obtained by a combined dye method fractionation of murine bone marrow

Blood ◽

10.1182/blood.v75.6.1240.1240 ◽

1990 ◽

Vol 75 (6) ◽

pp. 1240-1246 ◽

Cited By ~ 18

Author(s):

I McAlister ◽

NS Wolf ◽

ME Pietrzyk ◽

PS Rabinovitch ◽

G Priestley ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Light Scatter ◽

Isolated Cells ◽

Term Survival ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Separation Media

Abstract Hematopoietic stem cells were purified from murine bone marrow cells (BMC). Their characteristic density, size, internal complexity, Hoechst 33342 dye uptake, and wheat germ agglutinin (WGA) affinity were used to distinguish them from other cells in the bone marrow. BMC suspensions were centrifuged over Ficoll Lymphocyte Separation Media (Organon Teknika, Durham, NC; density 1.077 to 1.08). The lower-density cells were drawn off, stained with Hoechst and labeled with biotinylated WGA bound to streptavidin conjugated to phycoerythrin (WGA-B*A-PE) or with WGA conjugated to Texas Red. These cells were then analyzed and sorted by an Ortho Cytofluorograph 50-H cell sorter. The cells exhibiting medium to high forward light scatter, low to medium right angle light scatter, low Hoechst intensity, and high WGA affinity were selected. Sorted BMC (SBMC) were stained with Romanowsky-type stains for morphologic assay, and were assayed in lethally irradiated (LI) mice for their ability to produce colony-forming units in the spleen (CFU-S) and for their ability to produce survival. The spleen seeding factor for day 8 CFU-S upon retransplantation of the isolated cells was 0.1. The isolated cells were found to have consistent morphology, were enriched up to 135-fold as indicated by day 8 CFU-S assay, 195-fold as indicated by day 14 CFU-S assay, and 150 sorter-selected BMC were able to produce long-term survival in LI mice with retention of donor karyotype. When recipients of this first transplantation were themselves used as BMC donors, their number of day 8 and day 12 CFU-S were found to be reduced. However, 3 X 10(5) of their BMC provided 100% survival among secondary recipients. When the previously SBMC were competed after one transplantation against fresh nonsorted BMC in a mixed donor transplant, they showed the decline in hematopoietic potency normally seen in previously transplanted BMC. We conclude that the use of combinations of vital dyes for fluorescence-activated cell sorting (FACS) selection of survival-promoting murine hematopoietic stem cells provides results comparable with those produced by antibody- selected FACS and has the advantage of a method directly transferable to human BMC.

Download Full-text

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia

Blood ◽

10.1182/blood-2010-09-308262 ◽

2011 ◽

Vol 117 (14) ◽

pp. 3737-3747 ◽

Cited By ~ 20

Author(s):

Dirk Heckl ◽

Daniel C. Wicke ◽

Martijn H. Brugman ◽

Johann Meyer ◽

Axel Schambach ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Aplastic Anemia ◽

Bone Marrow Cells ◽

Specific Expression ◽

Hematopoietic Stem ◽

Egfp Reporter

AbstractThpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl−/−) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl−/− bone marrow cells into Mpl−/− mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl−/− cells had increased long-term repopulating potential, with a marked increase in lineage−Sca1+cKit+ cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage−Sca1+cKit+ cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Download Full-text

Hemangiogenic Role of ELF4 in Bone Marrow Post Myeloablation.

Blood ◽

10.1182/blood.v108.11.1783.1783 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1783-1783

Author(s):

Mariela Sivina ◽

Takeshi Yamada ◽

Natalie Dang ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Hematopoietic Stem Cells ◽

Blood Vessels ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Huvec Cells

Abstract Bone marrow suppression is an important cause of death in patients exposed to radiation or in cancer patients treated with conventional chemotherapeutic agents. Myeloablative treatments (i.e. 5-fluorouracil administration) lead to apoptosis of blood forming cells and to regression of blood vessels in bone marrow. It is well known that hematological recovery post-bone marrow insult depends on the capacity of hematopoietic stem cells to regenerate the entire hematopoietic system, however, the transcriptional machinery involved in the regeneration of sinusoidal blood vessels in bone marrow from endothelial progenitor cells is largely unknown. Endothelial cells express the Tie2 receptor tyrosine kinase (a.k.a. Tek), which is involved in the angiogenic remodeling and vessel stabilization. Gene targeting of Tie2 showed that it is not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo although Tie2 is needed during postnatal bone marrow hematopoiesis. ELF is a subgroup of the ETS family of transcription factors composed by ELF1, ELF2 (a.k.a. NERF), ELF3, ELF4 (a.k.a. MEF) and ELF5. ELF1 and ELF2 have been shown to regulate Tie2 expression in vitro. Recently we showed that ELF4 modulates the exit of hematopoietic stem cells (HSC) from quiescence (Lacorazza et al., Cancer Cell2006, 9:175–187). Given the high homology between ELF1 and ELF4 and the same origin of HSC and endothelial progenitor cells, we hypothesize that ELF4 regulates proliferation and Tie2 expression of endothelial cells. We used a luciferase gene reporter system in COS-7 and HEK cells to examine the capacity of ELF proteins to activate Tie2. ELF4 is the strongest activator of Tie2 expression following the hierarchy ELF4>ELF1>ELF2 variant 1>ELF2 variant 2. Site directed mutagenesis of each of the five ETS-binding sites (EBS) present in the Tie2 promoter shows that ELF4 binds preferentially to EBS 1, 3 and 5. Binding of ELF4 to the Tie2 promoter was confirmed by chromatin immunoprecipitation and EMSA. Although Elf1 gene expression is essentially normal in Elf4−/− bone marrow cells collected after 5-FU treatment, we detected diminished Tie2 expression compared to Elf4+/+ bone marrow cells. The association of this effect to human endothelial cells derived from umbilical cord (HUVEC cells) was investigated. All-trans retinoic acid (ATRA) and vascular-endothelial growth factor (VEGF) induced ELF4 expression in HUVEC cells in a dose and time dependent manner which was followed by increased Tie2 expression, suggesting that expression of ELF4 is modulated by angiogenic signals. Moreover, endothelial cells treated with ATRA showed rapid wound colonization in a wound assay. Expression of the pan-endothelial marker MECA-32 was determined by immunohistochemistry to correlate Tie2 with the regeneration of blood vessels: myeloablated Elf4−/− femurs exhibited a reduction of MECA-32 positive arterioles. Finally, temporal and spatial expression of Tie2 during hematological recovery post ablation was measured in bone marrow using transgenic Tie2-LacZ mice crossed to Elf4−/− mice. Collectively, our data suggests that ELF4 regulates Tie2 expression in endothelial cells but most importantly their proliferative capacity in response to angiogenic signals.

Download Full-text

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

Blood ◽

10.1182/blood.v114.22.2504.2504 ◽

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.

Download Full-text

Kruppel-Like Factor 10 (KLF10)-Deficient Mice Have Marked Defects In EPC Differentiation, Function, and Angiogenesis

Blood ◽

10.1182/blood.v116.21.4314.4314 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4314-4314

Author(s):

Akm Khyrul Wara ◽

Kevin Croce ◽

ShiYin Foo ◽

Xinghui Sun ◽

Basak Icli ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Hindlimb Ischemia ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Deficient Mice ◽

Tgf Β1 ◽

Impaired Angiogenesis

Abstract Abstract 4314 Background: Emerging evidence demonstrates that endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of being recruited to sites of ischemic injury and contribute to neovascularization. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Methods and Results: We previously identified that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) can preferentially differentiate into EPCs and possess high angiogenic activity under ischemic conditions compared to megakaryocyte-erythrocyte progenitors (MEPs), hematopoietic stem cells (HSCs), and common lymphoid progenitors (CLPs). Herein, we identify that a TGF-β1-responsive Kruppel-like Factor, KLF10, is robustly expressed in EPCs derived from CMPs and GMPs, compared to progenitors lacking EPC markers. KLF10–/– mice have marked defects in circulating EPCs (–23.6% vs. WT, P<0.004). In addition, EPC differentiation and TGF-β induced KDR responsiveness is markedly impaired (CMPs: WT 22.3% vs. KO 8.64%, P<0.0001; GMPs: WT 32.8% vs. KO 8.97%, P<0.00001). Functionally, KLF10–/– EPCs derived from CMPs and GMPs adhered less to fibronectin-coated plates (CMPs: WT 285 vs. KO 144.25, P< 0.0004; GMPs: WT 275.25 vs. KO 108.75, P <0.0003) and had decreased rates of migration in transwell Boyden chambers (CMPs: WT 692 vs. KO 298.66, P<0.00004; GMPs: WT 635.66 vs. KO 263.66, P<0.00001). KLF10–/– mice displayed impaired blood flow recovery after hindlimb ischemia (day 14, WT 0.827 vs. KO 0.640, P <0.009), an effect completely rescued by WT EPCs, but not KLF10–/– EPCs. Matrigel plug implantation studies demonstrated impaired angiogenesis in KLF10–/– mice compared to WT mice (WT 158 vs. KO 39.83, P<0.00000004). Overexpression studies revealed that KLF10 rescued EPC formation from TGF-β1+/– CMPs and GMPs. Mechanistically, TGF-β1 and KLF10 target the VEGFR2 promoter in EPCs which may underlie these effects. Background: Collectively, these observations identify that TGF-β1 signaling and KLF10 are part of a key signaling pathway that regulates EPC differentiation from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states. Disclosures: No relevant conflicts of interest to declare.

Download Full-text