VE Cadherin Positive Endothelial Cells Regulate Hematopoietic Reconstitution In Vivo.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3734-3734
Author(s):  
J. Lauren Russell ◽  
Phuong Doan ◽  
Heather A Himburg ◽  
Sarah K. Meadows ◽  
Pamela Daher ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Fold Increase ◽  
Small Subset ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution

Abstract Abstract 3734 We have recently demonstrated that targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ bone marrow endothelial cells (BM ECs) causes the protection of the BM sinusoidal vasculature, and the BM hematopoietic stem cell (BM HSC) pool following high dose total body irradiation (TBI). Since Tie2 is expressed on BM ECs and a small subset of quiescent BM HSCs, we developed a complementary model utilizing VE-cadherin Cre mice to more specifically confirm the function of BM ECs in regulating hematopoietic reconstitution in vivo. VE-cadherinCre;Bak−/−;BaxFl/− mice, which bear deletion of Bak and Bax in VE-cadherin+ ECs, and VE-cadherinCre;Bak−/−;BaxFl/+ control mice were generated and we compared the hematopoietic responses of these animals to high dose TBI. At steady state, these mice showed no difference in total BM cells, bone marrow ckit+sca+lineage- (KSL) progenitor cells, BM colony forming cells (CFCs), or BM colony forming unit spleen day 12 (CFU-S12) counts. At 2 hours after exposure to 500cGy TBI, the mice also did not demonstrate any significant differences in total BM cells, BM KSL cells, CFCs, or CFU-S12. However, 7 days after exposure to 500cGy TBI, VE-cadherin;Bak−/−;BaxFl/− mice displayed a 2-fold increase in total BM cells (p=0.006), a 3-fold increase in BM CFCs (p=0.0009), and 4-fold increase in BM CFU-S12 (p=0.079) compared to VE-cadherinCre;Bak−/−;BaxFl/+ control mice. Microscopic examination confirmed severe hypocellularity and corruption of the BM sinusoidal vasculature at day +7 post-TBI in VE-cadherinCre;Bak−/−;BaxFl/+ mice, whereas VE-cadherinCre;Bak−/−;BaxFl/− mice displayed nearly normal cellularity and preserved BM sinusoidal vessels. Taken together, these data demonstrate that VE-cadherin+ BM ECs regulate hematopoietic reconstitution in vivo and that targeted therapies aimed at augmentation of BM EC function can accelerate hematopoietic regeneration in vivo. Currently, we are comparing the cytokine production and gene expression profiles of VE-cadherinCre;Bak−/−;BaxFl/− BM ECs versus VE-cadherinCre;Bak−/−;BaxFl/+ BM ECs to identify candidate BM EC-derived proteins which are responsible for the protection of the BM stem/progenitor cell pool from radiation injury. VE-cadherin+ BM ECs represent an attractive mechanistic target for the identification of signaling pathways that regulate hematopoietic regeneration following injury. Disclosures: Chao: Genzyme: Research Funding.

Facilitation of Hematopoietic Reconstitution Via Inhibition of Bone Marrow Endothelial Cell-Mediated SDF-1 Signaling.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3859-3859
Author(s):  
Phuong L. Doan ◽  
J. Lauren Russell ◽  
Heather A. Himburg ◽  
Sarah K. Meadows ◽  
Pamela Daher ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
High Dose ◽  
Cell Recovery ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Hematopoietic Reconstitution

Abstract Abstract 3859 Hematopoietic stem cell (HSC) regeneration is influenced by specialized bone marrow (BM) microenvironments, but the mechanisms that drive HSC regeneration remain incompletely defined. We have recently reported that deletion of the pro-apoptotic proteins, Bak and Bax, in Tie2+ bone marrow endothelial cells (BM ECs)(Tie2Cre;Bak-/-;BaxFl/- mice) caused a significant protection of the BM HSC pool and the BM sinusoidal vasculature in mice following high dose total body irradiation (TBI). We also confirmed that this protection of the BM HSC pool was caused by protection of BM Tie2+ ECs via generation of chimeric mice (Tie2Cre;Bak-/-;BaxFl/- BM; wild type BM ECs) which contained 4.8-fold less BM long-term repopulating HSCs compared to mice bearing deletion of Bak and Bax in both BM HSCs and BM ECs. In order to determine the mechanism through which Tie2+ BM ECs regulate HSC regeneration, we generated primary BM EC lines from Tie2Cre;Bak-/-;BaxFl/- mice and Tie2Cre;Bak-/-;BaxFl/+ control mice. We then compared the capacity for Bak/Bax -/- BM ECs to support BM HSC regeneration in vitro compared to Bak/Bax +/&minus; BM ECs. BM c-kit+sca-1+lin- (KSL) stem/progenitor cells were irradiated with 300 cGy and then placed in 7 day culture with Bak/Bax -/- BM ECs or Bak/Bax +/&minus; BM ECs. Culture with Bak/Bax -/- BM ECs did not yield a significant increase in total viable cells, but yielded 2000-fold increased number of BM KSL cells (p < 0.05, n=3) compared to cultures with Bak/Bax +/&minus; ECs. This significant expansion of phenotypic BM stem/progenitor cells corresponded to a 4-fold increase in CFU-S12 cells in the Bak/Bax -/- EC cultures vs. Bak/Bax +/&minus; EC cultures (p=0.01, n=5). We subsequently compared the level of expression of several microenvironmental ligands which are putatively involved in regulating hematopoiesis. We found that BM ECs from Tie2Cre;Bak-/-;BaxFl/- mice had 37-fold lower expression of stromal-derived factor-1 (SDF-1, CXCL12) compared to BM ECs from Tie2Cre;Bak-/-;BaxFl/+ mice. Moreover, 7 days after TBI, Tie2Cre;Bak-/-;BaxFl/- mice had a 41-fold increase in total viable BM cell counts and had a persistently lower SDF-1 expression on BM ECs (2.7-fold) compared to Tie2Cre;Bak-/-;BaxFl/+ mice (p=0.003). Therefore, we hypothesized that inhibition of SDF-1 signaling might facilitate hematopoietic regeneration following injury. Interestingly, the addition of a blocking anti-SDF1 antibody to cultures of irradiated BM KSL cells with Bak/Bax -/- ECs caused a 50% increase in total cell recovery (p<0.05), a 2.5 fold increase in BM KSL cell recovery (p<0.05) and a 2.2-fold increase in BM CFC recovery (p<0.05) compared to culture with Bak/Bax -/- ECs alone. However, the addition of anti-SDF1 antibody caused a 3-fold decrease in CFU-S12 recovery compared to Bak/Bax -/- EC cultures without anti- SDF1 antibody (p<0.05). Taken together, these data suggest that inhibition of SDF-1 signaling via BM ECs accelerates BM progenitor cell regeneration following injury but is deleterious to the recovery of the BM HSC pool. Targeted therapies aimed at inhibition of SDF-1 signaling may facilitate short-term hematopoietic reconstitution following injury via modulation of BM vascular niche signaling, but this may be at the expense of the BM HSC pool. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Endothelial progenitor cell infusion induces hematopoietic stem cell reconstitution in vivo

Blood ◽  
2009 ◽  
Vol 113 (9) ◽  
pp. 2104-2107 ◽  
Author(s):  
Alice B. Salter ◽  
Sarah K. Meadows ◽  
Garrett G. Muramoto ◽  
Heather Himburg ◽  
Phuong Doan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Cells ◽  
Progenitor Cell ◽  
White Blood Cells ◽  
Fold Increase ◽  
Endothelial Progenitor ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution ◽  
Total Body

Hematopoietic stem cells (HSCs) reside in association with bone marrow (BM) sinusoidal vessels in vivo, but the function of BM endothelial cells (ECs) in regulating hematopoiesis is unclear. We hypothesized that hematopoietic regeneration following injury is regulated by BM ECs. BALB/c mice were treated with total body irradiation (TBI) and then infused with C57Bl6-derived endothelial progenitor cells (EPCs) to augment endogenous BM EC activity. TBI caused pronounced disruption of the BM vasculature, BM hypocellularity, ablation of HSCs, and pancytopenia in control mice, whereas irradiated, EPC-treated mice displayed accelerated recovery of BM sinusoidal vessels, BM cellularity, peripheral blood white blood cells (WBCs), neutrophils, and platelets, and a 4.4-fold increase in BM HSCs. Systemic administration of anti–VE-cadherin antibody significantly delayed hematologic recovery in both EPC-treated mice and irradiated, non–EPC-treated mice compared with irradiated controls. These data demonstrate that allogeneic EPC infusions can augment hematopoiesis and suggest a relationship between BM microvascular recovery and hematopoietic reconstitution in vivo.

Bone Marrow CD34+ Cells Expanded On Human Brain Endothelial Cells Reconstitutes Lethally Irradiated Baboons in a Variable Manner.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3214-3214
Author(s):  
Hiroto Araki ◽  
John Chute ◽  
Benjamin Petro ◽  
Naoyuki Katayama ◽  
Ronald Hoffman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Human Brain ◽  
Ex Vivo ◽  
Fold Increase ◽  
Biological Variation ◽  
Brain Endothelial Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells

Abstract Abstract 3214 Poster Board III-151 Increased cell dose has a positive impact on the therapeutic outcome of bone marrow (BM) transplantation. However, methods to successfully expand hematopoietic stem cells (HSC) from BM have yet to be achieved. It has been shown previously that ex vivo expansion of BM cells using porcine microvascular endothelial cells can rescue a baboon from a lethal dose of radiation (Brandt et al. Blood 1999). However, in prior studies baboons that received CD34+ cell doses less than 4 × 106 cells/kg body weight failed to achieve hematopoietic engraftment. In our current studies we have used human brain endothelial cells (HUBECs) and cytokines to expand BM cells and examined their ability to provide hematopoietic reconstitution in three lethally irradiated baboons following autologous transplantation. After ex vivo culture, the grafts represented a 1.8- to 2.1-fold expansion of CD34+ cells, a 3.7-fold to 13.2-fold increase of colony-forming cells (CFC), and a 1.9-fold to 3.2-fold increase of cobblestone area-forming cells (CAFC) in comparison to the input cell numbers. The animal (PA6873) which received expanded product of a suboptimal dose of CD34+ cells (1.6 × 106/kg) achieved only myeloid engraftment (day 24). Out of 3 baboons transplanted two displayed myeloid engraftment, one animal achieved both myeloid and platelet engraftment and the third animal (PA6888) failed to achieve engraftment. The animal (PA6893) which received the expanded product of 4.9 × 106/kg CD34+ cells achieved myeloid engraftment (WBC > 500/ml blood) by day 8 and platelet engraftment (>20,000/μl) by day 39. The WBC recovery of this baboon was comparable but the platelet recovery was delayed (31 day vs. 39 day) in comparison to that experienced by an animal that received a large number of unexpanded CD34+ cells (26 × 106 /kg). Interestingly, despite the grafts of all three animals having a similar degree of CD34+ cell expansion, similar progenitor cell (CFC, CAFC) expansion, a similar pattern of cell adhesion molecule expression and similar migration capacity across an SDF1 gradient, the hematopoietic reconstitution capacity of each graft differed greatly. Prior studies using human BM cells in non-contact HUBEC co-cultures in NOD/SCID mice demonstrated no such variability. Our current data indicates three possibilities for the variations in hematopoietic reconstitution observed in a baboon model. One possibility is that there might be cell autonomous biological variation of HUBEC expanded BM grafts. The second possibility is the expanded graft may have contained variable numbers of contaminating endothelial cells (HUBEC) contributing to variations in hematopoietic reconstitution. Transplantation of vascular endothelial cells without HSC rescue has been shown to enhance hematopoietic recovery following radiation in mice (Chute et al. Blood 2007). Biological variation amongst the hosts is unlikely since identical pre-transplant conditioning and post-transplant care was provided to all three baboons. In addition, since only non-adherent cells were harvested from the co-culture, it is possible that more primitive repopulating HSCs embedded within the endothelial monolayer might have been excluded from the graft. Taken together, our findings highlight inherent differences in the hematopoietic reconstitution capacity of expanded BM grafts in xenotransplantation studies and large animal models. Disclosures No relevant conflicts of interest to declare.

Nfi Genes Are Novel Regulators Of Murine Hematopoietic Stem- and Progenitor Cell Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 735-735
Author(s):  
Per Holmfeldt ◽  
Pardieck Jennifer ◽  
Shannon McKinney-Freeman
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Gene Family ◽  
Peripheral Blood ◽  
Fetal Liver ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Hematopoietic Stem ◽  
Post Transplant

Abstract Hematopoietic stem cells (HSCs) are responsible for life-long maintenance of hematopoiesis. HSC transplantation represents one of the most heavily exploited cell based therapies, routinely used to treat a myriad of life threating disorders, such as leukemia and bone marrow failure. Identifying the molecular pathways that regulate HSC engraftment is crucial to further improving outcomes in patients that rely on HSC transplantation as a curative therapy. By examining the global gene expression profiles of highly purified HSC (Lineage-Sca-1+c-Kit+CD150+CD48-), we recently identified the following members of the Nfi gene family of transcription factors as highly expressed by HSC (McKinney-Freeman et al., Cell Stem Cell, 2012): Nfix, Nfia, and Nfic. These data suggest that Nfi genes may play a novel role in regulating HSC function. To test this hypothesis, HSCs were enriched from adult bone marrow (Lineage-, c-kit+, Sca-1+ (LSK) cells) and then transduced, individually, with lentiviruses carrying shRNAs targeting each Nfi gene. Twenty-four hours post-transduction, cells were injected into lethally irradiated mice along with untransduced bone marrow LSK competitor cells congenic at the CD45 allele. The peripheral blood of recipient mice was then analyzed periodically over 16 weeks for engraftment of the Nfi-depleted cells. Although shRNA mediated knockdown of Nfi gene expression had no effect on the in vitro cell growth or viability of LSK cells, Nfi-depleted HSCs displayed a significant loss of short- and long-term in vivo hematopoietic repopulating activity. This was true for Nfia-, Nfic-, and Nfix-deficient HSC. While Nfia and Nfic are only expressed by bone marrow HSC, Nfix is highly expressed by both bone marrow and fetal liver HSC. When Nfix was depleted by shRNAs from LSK cells purified from E14.5 fetal liver, a similar loss in competitive repopulating potential was seen. Lineage analysis of peripheral blood of recipients showed no significant differences in the distribution of the major blood lineages derived from LSK cells transduced with Nfi-specific shRNAs compared to controls. When the bone marrow of recipients transplanted with Nfix- depleted cells was examined 4 and 16 weeks post-transplant, a general loss of all hematopoietic stem- and progenitor compartments examined was seen relative to control. Thus, the observed decrease in repopulating activity occurs at the level of HSCs and multipotent progenitors. To confirm an essential role for an Nfi gene family member in the regulation of HSC engraftment post-transplant, LSK cells were purified from Nfix fl/fl mice, transduced with lentiviral Cre recombinase and subsequently introduced into lethally irradiated recipients alongside congenic competitor cells. Like LSK transduced with Nfix-specific shRNAs, Nfix-/- LSK cells failed to repopulate the peripheral blood of recipient mice as efficiently as control and similar trends were detected in all stem- and progenitor cell populations examined. Time-course experiments immediately following transplantation revealed that Nfix-depleted LSK cells establish themselves in the marrow of recipient mice as efficiently as control at 5 days post-transplant, but thereafter exhausted rapidly. Examination 10 days post-transplant revealed a 5-fold increase in apoptosis specifically in the LSK compartment, but not in its differentiated progeny, in recipients transplanted with Nfix-depleted LSK cells compared to control. The increase in apoptosis was not associated with any apparent change in the cell cycle status of the LSK cells. These data suggest that Nfi genes are necessary for the survival of HSC post-transplantation. In an effort to identify the molecular pathways regulated by Nfi genes in HSC, we acquired the global gene expression profiles of Nfix-depleted HSC. In agreement with our observation that Nfix-deficient HSC displays elevated levels of apoptosis following transplantation in vivo, we observed a significant decrease in multiple genes known to be important for HSC survival, such as Erg, Mecom and Mpl, in Nfix-depleted HSC. In summary, we have for the first time established a role for the Nfi gene family in HSC biology, as evident by a decrease in bone marrow repopulating activity in Nfi-depleted HSCs. By dissecting the precise role of Nfi genes in HSC biology, we will glean insights that could improve our understanding of graft failure in clinical bone marrow transplantations. Disclosures: No relevant conflicts of interest to declare.

Systemic Administration of Pleiotrophin Induces Hematopoietic Stem Cell Regeneration In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1498-1498
Author(s):  
Heather A Himburg ◽  
Pamela Daher ◽  
Sarah Kristen Meadows ◽  
J. Lauren Russell ◽  
Phuong Doan ◽  
...  
Keyword(s):  
Stem Cell ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Fold Increase ◽  
Cell Regeneration ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution

Abstract Abstract 1498 Poster Board I-521 Significant progress has been made toward delineating the intrinsic and extrinsic signaling pathways that regulate hematopoietic stem cell (HSC) self-renewal. However, much less is known regarding the process of HSC regeneration or the extrinsic signals that regulate hematopoietic reconstitution following stress or injury. Elucidation of the microenvironmental signals which promote HSC regeneration in vivo would have important implications for the treatment of patients undergoing radiation therapy, chemotherapy and stem cell transplantation. We recently reported that pleiotrophin, a soluble heparin-binding growth factor, induced a 10-fold expansion of murine long-term repopulating HSCs in short term culture (Himburg et al. Blood (ASH Annual Meeting Abstracts), Nov 2008; 112: 78). Based on this observation, we hypothesized that PTN might also be a regenerative growth factor for HSCs. Here we tested the effect of systemic administration of PTN to non-irradiated and irradiated C57Bl6 mice to determine if PTN could promote HSC regeneration in vivo. C57Bl6 mice were irradiated with 700 cGy total body irradiation (TBI) followed by intraperitoneal administration of 2 μg PTN or saline x 7 days, followed by analysis of BM stem and progenitor cell content. Saline-treated mice demonstrated significant reductions in total BM cells, BM c-kit+sca-1+lin- (KSL) cells, colony forming cells (CFCs) and long term culture-initiating cells (LTC-ICs) compared to non-irradiated control mice. In contrast, PTN-treated mice demonstrated a 2.3-fold increase in total BM cells (p=0.03), a 5.6-fold increase in BM KSL stem/progenitor cells (p=0.04), a 2.9-fold increase in BM CFCs (p=0.004) and an 11-fold increase in LTC-ICs (p=0.03) compared to saline-treated mice. Moreover, competitive repopulating transplantation assays demonstrated that BM from PTN-treated, irradiated mice contained 5-fold increased competitive repopulating units (CRUs) compared to saline-treated, irradiated mice (p=0.04). Taken together, these data demonstrate that the administration of PTN induces BM HSC and progenitor cell regeneration in vivo following injury. Comparable increases in total BM cells, BM KSL cells and BM CFCs were also observed in PTN-treated mice compared to saline-treated controls following 300 cGy TBI, demonstrating that PTN is a potent growth factor for hematopoietic stem/progenitor cells in vivo at less than ablative doses of TBI. In order to determine whether PTN acted directly on BM HSCs to induce their proliferation and expansion in vivo, we exposed mice to BrDU in their drinking water x 7 days and compared the response to saline treatment versus PTN treatment. PTN-treated mice demonstrated a significant increase in BrDU+ BM KSL cells compared to saline-treated controls (p=0.04) and cell cycle analysis confirmed a significant increase in BM KSL cells in S phase in the PTN-treatment group compared to saline-treated controls (p=0.04). These data indicate that PTN serves as a soluble growth factor for BM HSCs and induces their proliferation and expansion in vivo while preserving their repopulating capacity. These results suggest that PTN has therapeutic potential as a novel growth factor to accelerate hematopoietic reconstitution in patients undergoing myelosuppressive radiotherapy or chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Gene Expression Profiling of Murine HOXB4-Transduced HSCs Shows Multiple Counter-Regulatory Signals That May Control HSC Pool Size

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2361-2361
Author(s):  
Hui Yu ◽  
Sheng Zhou ◽  
Geoffrey A. Neale ◽  
Brian P. Sorrentino
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Array ◽  
Self Renewal ◽  
Time Points ◽  
Time Point

Abstract Abstract 2361 HOXB4 is a homeobox transcription factor that can induce hematopoietic stem cell (HSC) expansion both in vivo and in vitro. An interesting feature of HOXB4-induced HSC expansion is that HSC numbers do not exceed normal levels in vivo due to an unexplained physiological capping mechanism. To gain further insight into HOXB4 regulatory signals, we transplanted mice with bone marrow cells that had been transduced with a MSCV-HOXB4-ires-YFP vector and analyzed gene expression profiles in HSC-enriched populations 20 weeks after transplant, a time point at which HSC numbers have expanded to normal levels but no longer increasing beyond physiologic levels. We used Affymetrix arrays to analyze gene expression profiles in bone marrow cells sorted for a Lin−Sca-1+c-Kit+ (LSK), YFP+ phenotype. Using ANOVA, we identified1985 probe sets with >2 fold difference in expression (FDR<, 0.1) relative to a control vector-transduced LSK cells. A cohort of genes was identified that were known positive regulators of HSC self-renewal and proliferation. Hemgn, which we identified in a previous screen as a positive regulator of expansion and a direct transcriptional target of HOXB4, was 3.5 fold up-regulated in HOXB4 transduced LSKs. Other genes known to be important for HSCs survival, self-renewal and differentiation were upregulated to significant levels including N-myc, Meis1, Hoxa9, Hoxa10 and GATA2. Microarray data for selected genes was validated by quantitative real-time PCR on HOXB4 transduced CD34low LSK cells, a highly purified HSC population, obtained from another set of transplanted mice at the 20 week time point. In contrast, other gene expression changes were noted that would potentially limit or decrease stem cell numbers. PRDM16, a set domain transcription factor critical for HSC maintenance and associated with clonal hematopoietic expansions when inadvertently activated as a result of retroviral insertion, was dramatically down-regulated on the expression array and 7.6 fold decreased in the real time PCR assay of CD34low LSK cells. TFG-beta signaling is a well defined inhibitor HSC proliferation and utilize Smad proteins as downstream effectors. Expression of Smad1 and Smad7 were significantly upregulated on the LSK expression array and 8.1 and 3.5 fold up-regulated by qPCR in CD34low LSK cells. Another potential counter-regulatory signal was down regulation of Bcl3 mRNA, a potential anti-apoptotic effector in HSCs. We hypothesize that the HOXB4 expansion program involves activation of genes that lead to increased HSC numbers with later activation of counter-regulatory signals that limit expansion to physiologic numbers of HSCs in vivo. We are now examining how this program changes at various time points after transplantation and hypothesize the capping limits are set at relatively later time points during reconstitution. We also are studying the functional effects of these gene expression changes, and in particular, whether enforced expression of HOXB4 and PRMD16 will result in uncontrolled HSC proliferation and/or leukemia. Disclosures: No relevant conflicts of interest to declare.

Disturbed Endothelial Blood-Bone Marrow-Barrier In Nox4 Deficient Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1169-1169
Author(s):  
Maren Weisser ◽  
Kerstin B. Kaufmann ◽  
Tomer Itkin ◽  
Linping Chen-Wichmann ◽  
Tsvee Lapidot ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Steady State ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Deficient Mice ◽  
Permeability State

Abstract Reactive oxygen species (ROS) have been implicated in the regulation of stemness of hematopoietic stem cells (HSC). HSC with long-term repopulating capabilities are characterized by low ROS levels, whereas increased ROS levels correlate with lineage specification and differentiation. Several tightly regulated sources of ROS production are well known among which are the NADPH oxidases (Nox). HSC are known to express Nox1, Nox2 and Nox4, however, their role in maintenance of stem cell potential or in the activation of differentiation programs are poorly understood. While Nox2 is activated in response to various extrinsic and intrinsic stimuli, mainly during infection and inflammation, Nox4 is constitutively active and is considered to be responsible for steady-state ROS production. Consequently, Nox4 deficiency might lower ROS levels at steady-state hematopoiesis and thereby could have an impact on HSC physiology. In this work we studied HSC homeostasis in Nox4 knock-out mice. Analysis of the hematopoietic stem and progenitor cell (HSPC) pool in the bone marrow (BM) revealed no significant differences in the levels of Lineage marker negative (Lin-) Sca-1+ ckit+ (LSK) and LSK-SLAM (LSK CD150+ CD48-) cells in Nox4 deficient mice compared to wild type (WT) C57BL/6J mice. HSPC frequency upon primary and secondary BM transplantation was comparable between Nox4 deficient and WT mice. In addition, the frequency of colony forming cells in the BM under steady-state conditions did not differ between both mouse groups. However, Nox4 deficient mice possess more functional HSCs as observed in in vivo competitive repopulating unit (CRU) assays. Lin- cells derived from Nox4 knock out (KO) mice showed an increased CRU frequency and superior multilineage engraftment upon secondary transplantation. Surprisingly, ROS levels in different HSPC subsets of NOX4 KO mice were comparable to WT cells, implying that the absence of Nox4 in HSCs does not have a major intrinsic impact on HSC physiology via ROS. Therefore, the increased levels of functional HSCs observed in our studies may suggest a contribution of the BM microenvironment to steady-state hematopoiesis in the BM of Nox4 KO animals. Recent observations suggest a regulation of the BM stem cell pool by BM endothelial cells, in particular by the permeability state of the blood-bone marrow-barrier (Itkin T et al., ASH Annual Meeting Abstracts, 2012). Endothelial cells interact with HSCs predominantly via paracrine effects and control stem cell retention, egress and homing as well as stem cell activation. As Nox4 is highly expressed in endothelial cells and is involved in angiogenesis, we reasoned that the absence of NOX4 could affect HSC homeostasis through altered BM endothelium properties and barrier permeability state. Indeed, in preliminary assays we found reduced short-term homing of BM mononuclear cells into the BM of Nox4 deficient mice as compared to wild type hosts. Furthermore, in vivo administration of Evans Blue dye revealed reduced dye penetration into Nox4-/- BM compared to wild type mice upon intravenous injection. Taken together, these data indicate a reduced endothelial permeability in Nox4 KO mice. Ongoing experiments aim at further characterization of the Nox4-/- phenotype in BM sinusoidal and arteriolar endothelial cells, the impact of Nox4 deletion on BM hematopoietic and mesenchymal stem cells, and in deciphering the role of Nox4 in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2488-2488 ◽  
Author(s):  
José Gabriel Barcia Durán
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Fluorescent Microscopy ◽  
Hematopoietic Stem ◽  
Interleukin Receptors ◽  
Sinusoidal Endothelium

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

Xenografts of Pediatric Acute Lymphoblastic Leukemia Retain the Gene Expression Pattern From the Diagnostic Material They Originated From Over Serial Passages.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1629-1629
Author(s):  
Manon Queudeville ◽  
Elena Vendramini ◽  
Marco Giordan ◽  
Sarah M. Eckhoff ◽  
Giuseppe Basso ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Leukemia Cells ◽  
Diagnostic Samples ◽  
Xenotransplantation Model

Abstract Abstract 1629 Poster Board I-655 Primary childhood acute lymphoblastic leukemia (ALL) samples are very difficult to culture in vitro and the currently available cell lines only poorly reflect the heterogeneous nature of the primary disease. Many groups therefore use mouse xenotransplantation models not only for in vivo testing but also as a means to amplify the number of leukemia cells to be used for various analysis. It remains unclear as to what extent the xenografted samples recapitulate their respective primary leukemia. It has been suggested for example that transplantation may result in the selection of a specific clone present only to a small amount in the primary diagnostic sample. We used a NOD/SCID xenotransplantation model and injected leukemia cells isolated from fresh primary diagnostic material of 4 pediatric ALL patients [2 pre-B-ALL, 1 pro-B-ALL (MLL/AF4}, 1 cortical T-ALL] intravenously into the lateral tail vein of unconditioned mice. As soon as the mice presented clinical signs of leukemia, leukemia cells were isolated from bone marrow and spleen. Isolated leukemia cells were retransplanted into secondary and tertiary recipients. RNA was isolated from diagnostic material and serial xenograft passages and gene expression profiles were obtained using a human whole genome array (Affymetrix U133 2.0). Simultaneously, immunophenotypic analysis via multicolor surface and cytoplasmatic staining by flow cytometry was performed for the diagnostic samples and respective serial xenograft passages. In an unsupervised clustering analysis the diagnostic sample of each patient clustered together with the 3 derived xenograft samples, although the 3 xenograft samples clustered stronger to each other than to their respective diagnostic sample. Comparison of the 4 diagnostic samples vs. all xenograft samples resulted in a gene list of 270 genes upregulated at diagnosis and 8 genes upregulated in the xenograft passages (Wilcoxon, p< .05). The high number of genes upregulated at diagnosis is most likely due to contamination of primary patient samples with normal peripheral blood and/or bone marrow cells as 15% of genes are attributed to myeloid cells, 7% to erythroid cells, 7% to lymphoid cells, 32% to bone marrow in general as well as to innate immunity, chemokines, immunoglobulins. The remaining genes can not be attributed to a specific hematopoetic cell lineage and are not known to be related to leukemia or cancer in general. Accordingly, there are no statistically significant differences between the primary, secondary and tertiary xenograft passages. The immunophenotype analysis are also in accordance with these findings, as the diagnostic blast population retains its immunophenotypic appearance during serial transplantation, whereas the contaminating CD45-positive non- leukemic cells disappear after the first xenograft passage. The few genes upregulated in xenograft samples compared to diagnosis are mainly involved in cell cycle regulation, protein translation and apoptosis resistance. Some of the identified genes have already been described in connection with cancer subtypes, their upregulation therefore being indicative of a high proliferative state in general and could argue towards a more aggressive potential of the engrafted leukemia cells but alternatively could also simply be due to the fact that the xenograft samples are pure leukemic blasts and are not contaminated with up to 15% of non-cycling healthy bone marrow cells as in the diagnostic samples. We conclude that the gene expression profiles generated from xenografted leukemias are very similar to those of their respective primary leukemia and moreover remain stable over serial retransplantation passages as we observed no statistically significant differences between the primary, secondary and tertiary xenografts. The differentially expressed genes between diagnosis and primary xenotransplant are most likely to be due to contaminating healthy cells in the diagnostic samples. Hence, the NOD/SCID-xenotransplantation model recapitulates the primary human leukemia in the mouse and is therefore an appropriate tool for in vivo and ex vivo studies of pediatric acute leukemia. Disclosures No relevant conflicts of interest to declare.

Tie2+ Bone Marrow Endothelial Cells Regulate Hematopoietic Reconstitution In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 248-248
Author(s):  
Phuong L. Doan ◽  
J. Lauren Russell ◽  
Sarah K. Meadows ◽  
Heather A Himburg ◽  
Pamela Daher ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Knockout Mice ◽  
Fold Increase ◽  
Chimeric Mice ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution ◽  
Total Body ◽  
Hematopoietic Response

Abstract Abstract 248 Radiation and chemotherapy cause myelosuppression in patients via damage to bone marrow (BM) hematopoietic stem cells (HSCs) and progenitor cells. It has been shown that BM HSCs reside in close association with osteoblasts and that BM osteoblasts regulate the maintenance of quiescent HSCs in vivo. HSCs also reside in proximity to BM sinusoidal vessels but the function of BM endothelial cells (BM ECs) in regulating HSC fate in vivo remains less well understood. We hypothesized that BM ECs play a critical role in regulating BM hematopoietic reconstitution following stress. In order to test this hypothesis, we utilized Cre-LoxP recombination to generate mice bearing targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ BM ECs (Tie2Cre;Bak−/− ;BaxFl/− mice) and measured their hematopoietic response to total body irradiation (TBI). Tie2Cre;Bak−/−;BaxFl/− mice (EC-Bak;Bax knockouts) were compared with Tie2Cre;Bak−/−;BaxFl/+ mice (EC-Bak;Bax (+) mice) which have constitutive Bak deletion but retain Bax in Tie2+ BM ECs. EC-Bak;Bax knockout mice had no detectable Bak or Bax by Western Blot in vascular tissues. After exposure to 100 cGy total body irradiation (TBI), EC-Bak;Bax knockout mice displayed a 2-fold increase in total viable BM cells (p=0.04), a 3-fold increase in BM ckit+sca+lineage- (KSL) progenitor cells, a 3-fold increase in colony-forming unit-spleen day 12 (CFU-S12) content (p=0.0003) and a 2-fold increase in 4 week competitive repopulating units (CRUs) compared to EC-Bak;Bax (+) mice. After 300 cGy TBI, comparable radioprotection was observed in the EC-Bak;Bax knockout mice, with a 1.9-fold increase in total BM cells, a 2.4-fold increase in CFU-S (p=0.01) and a 3.6-fold increase in 4 week CRU compared to EC-Bak;Bax (+) mice. Taken together, these results suggested that targeted protection of Tie2+ BM ECs from the intrinsic pathway of apoptosis mediated protection of BM hematopoietic stem/progenitor cells following TBI. Since Tie2 is expressed by BM ECs and a small subset of quiescent BM HSCs, we carried out experiments to determine whether the radioprotection we observed in EC-Bak;Bax knockout mice was caused autonomously by protection of Tie2+ BM ECs or Tie2+ HSCs. We transplanted 4 × 106 BM cells from EC-Bak;Bax knockout mice into lethally irradiated (950 cGy) wild type (WT) B6.SJL mice such that, after 12 weeks post-transplant, the recipient mice were chimeric for Bak and Bax deletions only in hematopoietic cells while retaining a wild type BM microenvironment (HSC-Bak;Bax knockout;EC-wild type), verified by qRTPCR. At 16 weeks post-transplant, the chimeric recipient mice were then exposed to 100 and 300 cGy TBI and we compared the hematopoietic response of these mice to that of EC-Bak;Bax knockout mice. Interestingly, after 100 cGy TBI, the hematopoietic response of the chimeric mice was comparable to that of wild type C57Bl6 mice and revealed a significant reduction in total viable BM cells (1.3-fold), BM KSL cells (7.4 fold) and BM CRU-4 weeks (9-fold) compared to 100 cGy-irradiated EC-Bak;Bax knockout mice. Significant reductions in total BM cells and BM CRU content were also observed in chimeric mice compared to EC-Bak;Bax knockout mice following 300 cGy TBI. These results suggest that deletion of Bak and Bax-mediated apoptosis in Tie2+ BM ECs protects BM hematopoietic stem and progenitor cells from ionizing radiation damage and this protection is largely autonomous to Tie2+ BM ECs. More generally, these data demonstrate that protection of BM ECs from the deleterious effects of ionizing radiation results in augmented hematopoietic reconstitution following TBI. This study demonstrates that Tie2+ BM ECs have an essential role in regulating hematopoietic regeneration following injury and reveals that BM ECs are a novel and attractive therapeutic target to augment hematopoietic reconstitution in vivo. Disclosures: No relevant conflicts of interest to declare.

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