scholarly journals CXCR4-SDF-1 Signaling in Nestin+ Mesenchymal Stem Cell Is Required for HSC Maintenance during Homeostasis and Regeneration after Irradiation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3883-3883 ◽  
Author(s):  
Pratibha Singh ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Stromal Cell ◽  
Cxcr4 Expression ◽  
Wild Type ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution

Hematopoietic stem cells (HSC) reside in a complex microenvironment (niche) within the bone marrow (BM), where multiple populations of microenvironmental stromal cells regulate and finely tune their proliferation, differentiation and trafficking. Recent studies have shown that mesenchymal stem cells (MSC) are an essential component of the HSC niche. Intrinsic HSC CXCR4-SDF-1 signaling has been implicated in self-renewal and quiescence; however, the role of microenvironment CXCR4-SDF-1 signaling in supporting HSC function remains unclear. We previously demonstrated that microenvironmental stromal cell-derived CXCR4 is important for HSC recovery, as transplantation of wild-type HSC into CXCR4 deficient recipients showed reduced HSC engraftment. In this study, we now show that CXCR4-SDF-1 signaling in nestin+ MSC regulates HSC maintenance under normal homeostatic conditions and promotes hematopoietic regeneration after irradiation. Multivariate flow cytometry analysis of marrow stroma cells revealed that mouse BM MSCs identified as CD45-Ter119-CD31-Nestin+PDGFR+CD51+ express the CXCR4 receptor, which was confirmed by RT-PCR analysis. To investigate the role of MSC CXCR4 signaling in niche maintenance and support of HSC function, we utilized genetic mouse models, in which CXCR4 could be deleted in specific stromal cell types. Selective deletion of CXCR4 from nestin+ MSC in adult tamoxifen inducible nestin-cre CXCR4flox/flox mice resulted in reduced total MSC in BM (Control vs. Deleted: 647±128 vs. 209±51/femur, respectively, n=5, p<0.05), which was associated with a significant reduction in Lineage-Sca-1+c-Kit+ (LSK) cells (Control vs. Deleted: 18,033±439 vs. 4523±358/femur, respectively n=5, p<0.05). Selective CXCR4 deletion in nestin+ MSC also resulted in enhanced LSK cell egress to the peripheral circulation (Control vs. Deleted: 1022±106 vs. 2690±757/ml blood, respectively n=5, p<0.05), with no detectable difference in HSC cell cycle or apoptosis. However, the repopulation ability of HSC obtained from mice where CXCR4 was deleted in nestin+ MSC was reduced by >2 fold. In contrast, deletion of CXCR4 from osteoblasts using osteocalcin cre CXCR4flox/flox mice had no effect on HSC numbers in BM and blood.To investigate the role of nestin+ MSC CXCR4 signaling in BM niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or nestin+ MSC CXCR4 deleted recipients after lethal irradiation (950 rad) and analyzed HSC homing, niche recovery and hematopoietic reconstitution. Deletion of CXCR4 from nestin expressing MSC resulted in significantly reduced LSK cell homing at 16 hrs post transplantation (Control vs. Deleted: 8643±1371 vs. 3004±1044/ mouse, respectively, n=5, p<0.05). Robust apoptosis and senescence after total body irradiation was observed in nestin expressing MSCs lacking CXCR4 expression. At 15 days post-transplantation, chimeric mice with nestin+ MSC lacking CXCR4 expression displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. In conclusion, our study suggests that CXCR4-SDF-1 signaling in nestin+ MSC is critical for the maintenance and retention of HSC in BM during homeostasis and promotes niche regeneration and hematopoietic recovery after transplantation. Furthermore, our data suggest the modulating CXCR4 signaling in the hematopoietic niche could be beneficial as a means to enhance HSC recovery following clinical hematopoietic transplantation or radiation/chemotherapy injury. Disclosures No relevant conflicts of interest to declare.

CXCR4-SDF-1 Signaling Mediated up-Regulation of Survivin Expression In Mesenchymal Progenitor Cells Is Critical for Their Proliferation and Maintenance of Osteoblastic Niche

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 941-941
Author(s):  
Pratibha Singh ◽  
Jennifer Speth ◽  
Peirong Hu ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Survivin Expression ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling

Abstract Abstract 941 Hematopoietic stem cells reside in osteoblastic and vascular niches within the bone marrow. The osteoblastic niche is composed of mesenchymal stem cell derived progenitor cells (MPC) and osteoblasts and are the main sources of the CXC chemokine CXCL12/SDF-1 in the bone marrow microenvironment. Several published studies suggest that the interaction between CXCR4 expressed on hematopoietic stem cells with SDF-1 produced in the bone marrow microenvironment is important for their retention in the bone-marrow. However, the role of SDF-CXCR4 signaling in formation and maintenance of osteoblastic niches in the bone marrow is not known. In this study, we examined the role of CXCR4 signaling in MPC proliferation and differentiation and its effects on hematopoietic stem cell (HSC) function. Flow cytometry analysis demonstrated that CXCR4 is expressed on the phenotypically defined MPC. Deletion of CXCR4 in tamoxifen cre inducible CXCR4flox-flox mice (verified by PCR and flow cytometry; 90% gene deletion and surface CXCR4 expression) results in significantly decreased numbers of Lin- CD45- CD31- Sca-1+ ALCAM- MPC (39±4.2%) and Lin- CD45- CD31- Sca-1-CD51+ osteoblasts (25±2.6%) in bone marrow 15 days after tamoxifen treatment. SDF-1 induced proliferation of CXCR4 deficient MPC was decreased by 4-fold compared to control, measured by the colony forming unit-fibroblast (CFU-F) assay. To determine, whether CXCR4 deficiency in bone marrow stromal cells affects SDF-1 induced HSC proliferation, we cultured FACS sorted wild-type SLAM SKL (103 cells) on CXCR4 deficient stroma for 5 days and total SLAM SKL cell numbers were counted by flow-cytometey analysis. CXCR4 deficient stroma failed to support optimal HSC proliferation and 48±5.2% less SLAM KSL cells was observed on CXCR4 deficient stroma compared to wild-type stroma. To investigate the mechanisms through which CXCR4-SDF-1 signaling regulates MPC proliferation, we evaluated the effect of SDF-1 treatment on expression of the anti-apoptotic and cell-cycle regulator protein, Survivin, in MPC. Multivariate intracellular flow cytometry demonstrated that Survivin expression increased by 23±4.2% in wild-type MPC after SDF-1 treatment (50ng/ml), however no significant increased was demonstrated in CXCR4 deficient MPC cells. CFU-F formation was reduced by 2.5 fold when the Survivin gene was conditionally deleted in MPC. Moreover, fewer SLAM SKL cells were detected on Survivin deficient stroma compared to wild-type stroma after SDF-1 treatment for 5 days. In conclusion, our data suggest that CXCR4-SDF-1 signaling mediated Survivin expression in MPC is important for their proliferation and maintenance of the bone-marrow hematopoietic niche. Disclosures: No relevant conflicts of interest to declare.

Evidence for the Involvement of CXCR4 Signaling in In Vivo Self-Renewal of Transplanted Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1888-1888 ◽  
Author(s):  
Chen-YI LAI ◽  
Makoto Otsu ◽  
Motohito Okabe ◽  
Sachie Suzuki ◽  
Satoshi Yamazaki ◽  
...  
Keyword(s):  
Post Transplantation ◽  
Gain Of Function ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Hematopoietic Reconstitution

Abstract Abstract 1888 Hematopoietic stem cells (HSCs) represent the unique cell population capable of self-renewal and multi-lineage differentiation, thereby lifelong sustainment of the hematopoiesis. HSC transplantation has proven beneficial for various diseases, it is therefore important to elucidate the molecular determinants for successful HSC engraftment. Signaling through the chemokine receptor CXCR4 has been implicated in HSC engraftment by the observation that transplantation of HSCs lacking this molecule results in poor hematopoietic reconstitution. Because this impairment, however, can be attributed to the defects in any of the post-transplantation processes that include bone marrow (BM)-homing, -repopulation, or –retention, it is still unclear whether CXCR4 plays an essential role in HSC self-renewal upon transplantation. To elucidate the role of CXCR4 signaling in HSC self-renewal in conjunction with transplantation, we used a purified CD34neg/low c-Kit+ Sca-1+ Lineage-markerneg population as the defined stem cell source. As a loss-of-function study, CXCR4 was conditionally deleted in HSCs before transplantation. As a gain-of-function study, we generated the HSC populations overexpressing either wild-type (wt)- or C-terminal truncated (δC)-CXCR4 (OE-HSCs), the latter of which is known to exhibit enhancement in the SDF-1 signaling, by gene transfer and subsequent cell sorting. We compared these cells with control HSCs in in vitro assays with regard to the biological characteristics including chemotaxis, proliferation, colony formation, and cobblestone-area (CA) forming ability. To dissect in vivo post-transplantation processes, we investigated hematopoietic repopulation kinetics in the recipient BM at the homing/lodging phase (within 1 wk) and the early repopulation phase (2–3 wks) for the above test HSCs. The self-renewal potential of each HSC population was estimated by competitive repopulation assay. In vitro studies: OE-HSCs with wt- or δC-CXCR4 exhibited enhanced chemotaxis and proliferation in response to SDF1, confirming the gain-of-function effects of these modifications. CA forming ability was greater in OE-HSCs with δC-CXCR4 than control counterparts and absent in CXCR4-KO HSCs, suggesting the critical role of CXCR4-signaling in HSC proliferation in the presence of stromal support. In vivo studies: 1) the homing/lodging phase. Unexpectedly, we did not find significant alteration in the numbers of early progenies detectable in recipient BM 3 days after transplantation of HSCs receiving either loss- or gain-of-function modification to CXCR4, indicating that this signaling is indispensable in HSC homing. 2) the early repopulation phase. Impairment of hematopoietic repopulation in BM became evident for CXCR4-KO HSCs through 2–3 wks. On the other hand, OE-HSCs with CXCR4, more remarkably of ΔC-mutation, showed enhanced BM repopulation kinetics at ∼3 wks post transplantation, suggesting the importance of CXCR4 signaling in HSC amplification at this post-transplantation phase. 3) long-term hematopoiesis. CXCR4-KO-HSCs showed poor hematopoietic reconstitution potentials, consistent with previous observations. Interestingly, impaired peripheral repopulation was also observed with OE-HSCs with wt- or ΔC-CXCR4. Further characterization revealed that the recipients of CXCR4-overexpressing HSCs did retain their progenies, which showed multilineage differentiation, but exhibited impaired release of mature leukocytes from the BM to the peripheral blood. Most importantly, however, test-cell chimerism in the long-term HSC fraction was significantly higher in the mice receiving OE-HSCs with CXCR4, especially of ΔC-type, than those transplanted with control HSCs, indicating that the augmentation of CXCR4 signaling enhanced competitive repopulation ability of HSCs. These modified HSCs demonstrated repopulation abilities also in secondary recipients. We demonstrated that CXCR4 signaling is indispensible for HSC homing and that continuous overexpression of CXCR4 cannot benefit the peripheral reconstitution in contrary to the expectation. More importantly, our studies showed that augmentation of CXCR4 signaling leads to HSC expansion in vivo upon transplantation. We thus conclude that CXCR4 signaling has a role in HSC self-renewal and that its regulation may find the approach that will improve HSC transplantation outcomes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3758-3779 ◽  
Author(s):  
N Uchida ◽  
HL Aguila ◽  
WH Fleming ◽  
L Jerabek ◽  
IL Weissman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Critical Role ◽  
White Blood Cells ◽  
Cell Types ◽  
Dose Dependence ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery

Abstract Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca- 1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.

scholarly journals The Role of NADPH Oxidase 2 in Normal and Malignant Hematopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1079-1079
Author(s):  
Biniam Adane ◽  
Haobin Ye ◽  
Shanshan Pei ◽  
Nabilah Khan ◽  
Mohammad Minhajuddin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Advisory Board ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Knock Out

Abstract NADPH dependent oxidase 2 (NOX2) is the founding member of a family of multimeric, oxido-reductase enzymes that catalyze the production of superoxides by transferring a single electron from the cofactor NADPH to molecular oxygen. It is primarily utilized in neutrophils and macrophages to generate copious amount of reactive oxygen species (ROS) to facilitate the neutralization of engulfed particulates during phagocytosis. In sharp contrast to this specialized function however, recent evidence implies a non-phagocytic role for NADPH oxidases in which physiologic levels of ROS generated by these enzymes modulate key signaling proteins and transcription factors to exert profound biological effects. Based on this information we decided to investigate the potential role of NOX2 in normal and leukemic stem cells. Using transgenic NOX2 knock out mice, genetically defined murine models of myeloid leukemia and primary human acute myeloid leukemia (AML) specimens, we show that NOX2 is critical for the proper function of normal and malignant hematopoietic stem cells. In silico analysis using published transcriptional profiles of hematopoietic populations revealed that multiple subunits of the NOX2 complex are expressed at low levels in hematopoietic stem cells (HSCs) and at relatively higher levels in multipotent progenitors (MPPs). Next, we characterized the different hematopoietic compartments from age and sex matched wild type (WT) and transgenic NOX2 knock out (KO) mice. Our studies revealed that in the bone marrow of KO mice, a subset of multipotent progenitor populations (MPP2 & MPP3), which often have biased myelo-erythroid output are markedly expanded relative to their wild type counterparts. Consistently, we found increased levels of granulocytes and monocytes in the peripheral circulation of NOX2 KO mice. To test whether NOX2 has a functional, biological role in the self-renewal of HSCs, we performed competitive transplantation assays using equal numbers of whole BM cells from WT and KO mice to co-repopulate lethally irradiated hosts. Analysis of engrafted mice showed that the contribution from NOX2 KO HSCs was severely compromised in all lineages and developmental stages of hematopoiesis examined. Collectively, these results suggest a critical biological role for NOX2 in maintaining the quiescence and long term self-renewal of HSCs. Similar to normal hematopoiesis, we found out that NOX2 is also widely expressed by functionally defined leukemic stem cells in a murine model of myeloid leukemia generated by expressing the oncogenic translocations BCR-ABL and NUP98-HOXA9. To evaluate the role of NOX2 in leukemogenesis, we established the BCR-ABL/NUP98-HOXA9 model using primitive cells derived from either WT or KO. Intriguingly, NOX2 KO leukemic cells generated a much less aggressive disease upon transplantation into primary and subsequently into secondary recipients. Furthermore, leukemic cells in which NOX2 is suppressed displayed aberrant mitotic activity and altered developmental potential marked by loss of quiescence, enhanced entry into cycle and terminal differentiation. To gain mechanistic insight into the observed phenotype, we isolated leukemic stem cells and performed whole genome expression analysis. The data showed that deficiency of NOX2 leads to downregulation of the cell cycle inhibitor CDKN2C (p18) and robust activation of the granulocyte fate determining transcription factor CEBPε. Thus we conclude that loss of NOX2 impacts leukemogenesis through rewiring of the cell cycle machinery and developmental programs in leukemic stem cells. Finally, we found that in CD34+ primary human AML cells, NOX2 and the other subunits of the complex are abundantly expressed. Furthermore, pharmacologic inhibition of NOX2 with VAS2870, a selective NADPH oxidase inhibitor, reduced the level of ROS and limited the in vitro proliferation and survival of leukemic cells. Next we genetically suppressed the expression of NOX2 in primary human AML cells using sh-RNAs and transplanted these cells into immune compromised mice. Consistent with the murine leukemia, NOX2 knocked down AML cells failed to engraft and expand in vivo. Taken together, our results firmly establish a hitherto unrecognized, prominent regulatory role for NOX2 in the biology of normal and malignant hematopoietic stem cells and imply a potential therapeutic opportunity that can get exploited to treat AML. Disclosures Pollyea: Celgene: Other: advisory board, Research Funding; Ariad: Other: advisory board; Pfizer: Other: advisory board, Research Funding; Glycomimetics: Other: DSMB member; Alexion: Other: advisory board.

Loss of Gfi1 Impedes Development of T-Cell Lymphoma upon Exposure to N-ethyl-N-nitrosourea but Predisposes to Severe Myleodysplastic Changes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2221-2221
Author(s):  
Cyrus Khandanpour ◽  
Ulrich Duehrsen ◽  
Tarik Möröy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Cell Lymphoma ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Exogenous toxic substances often cause the initiation and development of leukemia and lymphoma by acting as mutagens. N-ethyl-N-nitrosourea (ENU) is a paradigmatic example for such a substance, which introduces point mutations in the genome through DNA damage and repair pathways. ENU is widely used to experimentally induce T-cell lymphomas in mice. We have used ENU to investigate whether the hematopoietic transcription factor Gfi1 is required for lymphomagenesis. The Gfi1 gene was originally discovered as a proviral target gene and a series of experiments with transgenic mice had suggested a role of Gfi1 as a dominant oncogene with the ability to cooperate with Myc and Pim genes in the generation of T-cell lymphoma. In addition, Gfi1 deficient mice showed a defect in T-cell maturation but also aberration in myeloid differentiation and an accumulation of myelomonocytic cells. ENU was administered i.p. once a week for three weeks with a total dose of 300mg/kg to wild type (wt) and Gfi1 null mice. Wild type mice (12/12) predominantly developed T-cell tumors and rarely acute myeloid leukemia, as expected. However, only 2/8 Gfi1 −/− mice succumbed to lymphoid neoplasia; they rather showed a severe dysplasia of the bone marrow that was more pronounced than in wt controls. These changes in Gfi1 null mice were accompanied by a dramatic decrease of the LSK (Lin-, Sca1- and c-Kit+) bone marrow fraction that contains hematopoietic stem cells and by a higher percentage (18%) of bone marrow cells, not expressing any lineage markers (CD4, CD 8, Ter 119, Mac1, Gr1, B220, CD3). In particular, we found that the LSK subpopulation of Gfi1 deficient mice showed a noticeable increase in cells undergoing apoptosis suggesting a role of Gfi1 in hematopoietic stem cell survival. In addition, Gfi1−/− bone marrow cells and thymic T-cells were more sensitive to DNA damage such as radiation and exposure to ENU than their wt counterparts pointing to a role of Gfi1 in DNA damage response. Our results indicate that Gfi1 is required for development of T-cell tumors and that a loss of Gfi1 may sensitize hematopoietic cells and possibly hematopoietic stem cells for programmed cell death. Further studies have to show whether interfering with Gfi1 expression or function might represent a tool in the therapy of leukemia.

IGF Binding Protein 2 Supports the Cycling of Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1604-1604
Author(s):  
HoangDinh Huynh ◽  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Ex Vivo ◽  
Bone Marrow Stroma ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
C Terminus ◽  
Marrow Stroma ◽  
Igf Signaling

Abstract Abstract 1604 Previously we identified IGFBP2 as an extrinsic factor that supports ex vivo expansion of hematopoietic stem cells (HSCs). The role of IGFBP2 in HSCs and cancer is very intriguing. IGFBP2 can bind to insulin-like growth factor (IGF) ligands and displays IGF-dependent growth inhibitory effects on many cell types. On the other hand, IGFBP2 is capable of stimulating growth of certain cancer cells, and is overexpressed in many cancer patients and its expression is correlated with cancer progression. Here we sought to study the role of IGFBP2 in regulation of the activity of normal HSCs. We showed that IGFBP2 was expressed in differentiated hematopoietic cells and bone marrow stroma but not in HSCs. Consistent with its gene expression pattern, IGFBP2-/- HSCs had similar repopulation activity as their wild-type counterparts. By contrast, when we transplanted HSCs into IGFBP2-/- or wild-type recipient mice, we found decreased in vivo repopulation of HSCs in primary and secondary transplanted IGFBP2-/- recipients, suggesting that the environmental IGFBP2 positively supports HSC activity. Further co-culture of HSCs with IGFBP2-/- or wild-type bone marrow stromal cells indicated that IGFBP2 produced by bone marrow stroma indeed supports HSC expansion. Consistently, HSCs in IGFBP2-/- mice showed decreased frequency and cell cycling, and had upregulated expression of cell cycle inhibitors of p21, p16, and p19. To determine whether IGFBP2's effect on HSCs depends on IGF signaling, we compared the repopulation of donor cells deficient for the IGF type I receptor in wild-type and IGFBP2-/- recipients. These HSCs that are defective in IGF signaling still have decreased repopulation in IGFBP2-/- recipients, suggesting that the environmental effect of IGFBP2 on HSCs is independent of IGF signaling. To identify the functional domain of IGFBP2 in regulation of HSC activity, we constructed IGFBP2 with mutated RGD domain or deleted c-terminus and used the mutant IGFBP2 proteins in ex vivo culture of HSCs. We found that the c-terminus of IGFBP2 is essential to support HSC activity. We are currently in the process of identifying the potential receptor of IGFBP2 on HSCs. In summary, we found that IGFBP2 supports the cycling of normal HSCs, and this effect is independent of IGF signaling. Our study is important in revealing the relationship among environmental cues and cell fates of stem cells and opens up a new avenue in investigation of the roles of IGFBP2 in stem cells and cancer. Disclosures: No relevant conflicts of interest to declare.

An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2333-2333
Author(s):  
Brian D. Adams ◽  
Shangqin Guo ◽  
Haitao Bai ◽  
Changchun Xiao ◽  
E. Premkumar Reddy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

Role Of Connexin 32 In Hematopoiesis: Maintaining Quiescence Of Hematopoietic Stem Cells and Their Proliferation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1185-1185
Author(s):  
Yoko Hirabayashi ◽  
Isao Tsuboi ◽  
Byung-Il Yoon ◽  
Jun Kanno ◽  
James Trosko ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Average Percentage ◽  
Type Group ◽  
Donor Cells ◽  
In The Wild

Abstract Previously, we reported the findings of our studies on the role of connexin (Cx) 32 during steady-state hematopoiesis and its potential protective role against leukemogenesis. Namely, in wild-type mice, Cx32 expression was solely detected in primitive hematopoietic stem/progenitor cells (HSCs/HPCs). Since Cxs are essential molecules for multicellular organisms, Cxs are surmised to be present in the hematopoietic tissue to facilitate cell-cell communication between HSCs/HPCs themselves rather than between HSCs/HPCs and stromal cells. In addition, Cx32-knockout (KO) mice showed the following characteristics: first, a prominent decrease in the number of peripheral mononuclear cells (PMCs) associated with various HPCs; second, a significant increase in the number of HSCs, at least until 20 weeks of age; and third, an apparently delayed regeneration of HPCs after chemical abrasion. Furthermore, the incidence of leukemogenicity induced by methylnitrosourea increased prominently. These possible leukemogenic propensities taken together imply that Cx32 plays an important role in maintaining steady-state hematopoiesis and in suppressing leukemogenesis. In this study, first, we examined the cell kinetics of HPCs [CFU-GM, colony forming unit (CFU) granulomacrophage; CFU-S9/CFU-S13, CFU in spleen on day 9/13] by evaluating the percentage of cycling HPCs with continuous incorporation of bromodeoxyuridine (BrdUrd) in vivo for up to 3 months, followed by exposure to ultraviolet-A to eliminate cells that incorporated BrdUrd. Using this method with the continuous incorporation of BrdUrd in vivo in mice up to 1.5 years of age, we discovered the existence of a long-term and stable, dormant fraction in the HPCs of wild-type mice. Without Cx32, the percentage of the entire cycling fraction of CFU-S13 apparently increased continuously, which indicates that Cx32 could restore the quiescence of hematopoiesis and thereby maintain HSCs/HPCs. This is consistent with the findings that the number of HPCs increased and the number of HSCs decreased with aging in Cx32-KO mice. Next, we examined the bone-marrow reconstitution capability of HSCs of Cx32-KO mice by serial transplantation. Five hundred cells in the lineage-negative, c-kit-positive, and Sca1-positive (LKS) fraction isolated from wild-type and Cx32-KO mice (Ly5.2) were transplanted into lethally irradiated first recipients (Ly5.1) separately with 2x105 freshly isolated bone marrow cells from wild-type F1(Ly5.1/Ly5.2) mice as rescuing cells, which prevent acute radiation injury. Two months after transplantation, both groups showed reconstituted hematopoiesis without any significant differences in various hematopoietic parameters, although mice reconstituted with cells in the LKS fraction without Cx32 showed a rather higher average percentage of donor-origin PMCs and a lower average percentage of the donor-origin cells in the LKS fraction than those with Cx32 (wild-type group vs. Cx32-KO group, average percentage of Ly5.2 with respect to the total ± standard deviations (s.d.); PMCs, 34.7±14.3% vs. 50.7±9.9 %, p=0.006; cells in the LKS fraction, 20.8±5.2% vs. 14.2±5.3%, p=0.110). Then, donor cells (Ly5.2) in the LKS fraction were isolated from the primary recipients of both groups separately and 400 cells were transferred into each secondary recipient (Ly5.1) with 2x105 rescuing cells from F1(Ly5.1/Ly5.2) mice. Although secondary recipients from both groups showed reconstituted hematopoiesis without any significant differences in various hematopoietic parameters four months after reconstitution, similar to the primary recipients, cells of donor origin in the LKS fraction could be detected only in the wild-type group. Namely, four out of seven recipients in the wild-type group showed over 0.5% donor cells and the average percentage and s.d. for 4 mice was 25.1±27.9%, whereas none of the recipients out of five in the Cx32-KO group showed more than 0.5%. The above-mentioned findings in this study in addition to the previous findings imply that Cx32 plays an essential role in maintaining self-renewal proliferation of primitive hematopoietic stem cells to prevent their exhaustion, and also in suppressing neoplastic changes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The staying power of hematopoietic stem cells

2021 ◽  
Vol 220 (11) ◽  
Author(s):  
Michael L. Dustin
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Stem And Progenitor Cells ◽  
Necessary And Sufficient

Hematopoietic stem and progenitor cells (HSPCs) use specialized adhesive structures referred to as magnupodium to stay in hematopoietic niches. Bessey et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202005085) define new characteristics of the magnupodium, including centriole polarization and the necessary and sufficient role of CXCR4 signaling.

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