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.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

Homing and Engraftment of Mobilized Hematopoietic Stem Cells: Involvement of VLA-5.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4943-4943
Author(s):  
Pieter K. Wierenga ◽  
Gerald de Haan ◽  
Bert Dontje ◽  
Ellen Weersing ◽  
Ronald van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Hematopoietic Reconstitution

Abstract VLA-5 has been implicated in the adhesive interactions of stem and progenitor cells with the bone marrow extracellular matrix and stromal cells and is therefore considered to play an important role in the hematopoietic reconstitution after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3% of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-GSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 38±3%. Despite this low frequency of VLA-5+ cells, however, even when equal numbers of progenitor cells are transplanted MPB cells provide a much faster hematopoietic recovery compared to BM cells. To shed more light on the role of VLA-5 in the process of homing and engraftment, we investigated whether differences in homing potential of the stem cell subsets might be responsible for this enhanced reconstitution. At 3 hours post-transplant, however, no differences in homing efficiency of progenitor and stem cells from MPB and BM grafts in both bone marrow and spleen could be detected. It should be realized that MPB and BM grafts demonstrate different ratios of stem/progenitor cells which might be another explanation for the observed differences in repopulation potential. Furthermore, MPB cells migrating in vitro towards SDF-1α showed potent reconstitution while VLA-5 expression was reduced on these cells. In fact, in vitro treatment with SDF-1α showed further decrease in VLA-5 expressing cells (from 38% to 4%) in the lin- fraction. When equal numbers of MPB were transplanted with and without SDF-1α pretreatment, no difference in hematopoietic reconstitution was observed suggesting a minor role of VLA-5 in homing and engraftment. On the other hand, after VLA-5 blocking an inhibition of 59±7% in the homing of MPB progenitor cells in the bone marrow could be found, whereas homing in the spleen of the the recipients is only inhibited by 11±4%. To elucidate whether the observed enhanced reconstitution could be explained by a selective homing of VLA-5+ cells or a rapid upregulation of VLA-5 expression, cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. It could be demonstrated that at 3 hours post-transplant cells from MPB grafts showed a rapid increase from 38±3% up to 66±9% of VLA-5+ cells in the bone marrow of the recipient. In the spleen no significant increase in VLA-5+ cells was observed. When MPB cells were transplanted after pretreatment with SDF-1α an increase from 2±1% up to 33±5% of VLA-5+ cells in the bone marrow was detected. When calculating the number of cells recovered from bone marrow, a selective homing of VLA-5+ cells cannot be excluded. Therefore, we also assessed the number of VLA-5+ cells in the PKH+ fraction in peripheral blood from the recipient immediately (½-1 hour) after transplantation but found no increase during that time period. So far it can be concluded that MPB cells show low number of VLA-5+ cells but these cells possess an enhanced hematopoietic reconstitution potential. Homing of progenitor cells to the spleen seems to be less dependent on VLA-5 expression than homing to the bone marrow. A rapid upregulation of VLA-5 expression on engrafting MPB cells early after transplantation does not occur and hence our data are suggestive for the preferential homing of VLA-5+ cells in the bone marrow after transplantation.

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.

Abstract 13: Plasminogen Is Required for Hematopoietic Stem Cell-Mediated Cardiac Repair After Myocardial Infarction

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Yanqing Gong ◽  
Jane Hoover-Plow ◽  
Ying Li
Keyword(s):  
Myocardial Infarction ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tissue Repair ◽  
Critical Role ◽  
Cardiac Repair ◽  
Internal Diameter ◽  
Hematopoietic Stem

Ischemic heart disease, including myocardial infarction (MI), is the primary cause of death throughout the US. Granulocyte colony-stimulating factor (G-CSF) is used to mobilize hematopoietic progenitor and stem cells (HPSC) to improve cardiac recovery after MI. However, poor-mobilization to G-CSF is observed in 25% of patients and 10-20% of healthy donors. Therefore, a better understanding of the underlying mechanisms regulating G-CSF-induced cardiac repair may offer novel approaches for strengthening stem cell-mediated therapeutics. Our previous studies have identified an essential role of Plg in HPSC mobilization from bone marrow (BM) in response to G-CSF. Here, we investigate the role of Plg in G-CSF-stimulated cardiac repair after MI. Our data show that G-CSF significantly improves cardiac tissue repair including increasing neovascularization in the infarct area, and improving ejection fraction and LV internal diameter by echocardiogram in wild-type mice. No improvement in tissue repair and heart function by G-CSF is observed in Plg -/- mice, indicating that Plg is required for G-CSF-regulated cardiac repair after MI. To investigate whether Plg regulates HPSC recruitment to ischemia area, bone marrow transplantion (BMT) with EGFP-expressing BM cells was performed to visualize BM-derived stem cells in infarcted tissue. Our data show that G-CSF dramatically increases recruitment of GFP+ cells (by 16 fold) in WT mice but not in Plg -/- mice, suggesting that Plg is essential for HPSC recruitment from BM to the lesion sites after MI. In further studies, we investigated the role of Plg in the regulation of SDF-1/CXCR-4 axis, a major regulator for HPSC recruitment. Our results show that G-CSF significantly increases CXCR-4 expression in infarcted area in WT mice. While G-CSF-induced CXCR-4 expression is markedly decreased (80%) in Plg -/- mice, suggesting Plg may regulate CXCR-4 expression during HSPC recruitment to injured heart. Interestingly, Plg does not affect SDF-1 expression in response to G-CSF treatment. Taken together, our findings have identified a critical role of Plg in HSPC recruitment to the lesion site and subsequent tissue repair after MI. Thus, targeting Plg may offer a new therapeutic strategy to improve G-CSF-mediated cardiac repair after MI.

The “MarrowMiner”: A Novel, Minimally Invasive Device for the Harvest of Bone Marrow: Initial Human Experience Reveals Safety, Efficacy and Richer Cell Product Than Standard Harvest Methods

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4130-4130
Author(s):  
Daniel L. Kraft ◽  
Vartan Ghazarossian ◽  
Mike Crocker ◽  
Sergio Najar ◽  
Antonio A. Carrasco-Yalðn
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
T Cell ◽  
Minimally Invasive ◽  
Progenitor Cells ◽  
Local Anesthesia ◽  
Peripheral Blood ◽  
Single Entry

Abstract INTRODUCTION: Bone marrow (BM) contains a rich supply of adult stem and progenitor cells, including hematopoieitic and mesenchymal stem cells which are used in Bone Marrow Transplantation (BMT) and an increasing array of regenerative therapies. Traditional marrow harvest methods utilize percutaneous large bore needle aspiration, result in marrow highly diluted by peripheral blood, and are crude, tedious, labor intensive and expensive, usually requiring general anesthesia, and &gt;100 serial small volume aspirates to obtain adequate cell numbers for BMT. BM is showing increasing long-term advantages over mobilized PBSC for many alloegeneic BMTs, in terms of less cGVHD and in some cases improved survival. Improved BM harvest methods are needed. A novel device, the “MarrowMiner” (MM), was developed for the minimally invasive harvest of BM to enable the rapid, convenient, outpatient harvest of large quantities of BM under local anesthesia for use in allogeneic and autologous BMT and cell therapies utilizing autologous marrow derived cells. The MarrowMiner utilizes a single marrow entry site into the anterior or posterior iliac, through which the flexible, powered, guidable FlexShaft catheter can access the majority of the marrow space and aspirate rich marrow. Extensive testing in human cadavers and porcine models demonstrated a 10X increase in stem cells activity/ml (by CFU) compared to that of traditional needle harvests. The MM recently received both FDA and CE Mark regulatory approved, and ‘First In Human’ trials were successfully completed under local anesthesia, demonstrating safety, efficacy and higher stem cell yields compared to traditional methods. METHODS: In an ongoing prospective study, 10 patients undergoing autologous marrow derived therapy for use in regenerative medicine, had marrow harvested from their anterior or posterior ileac by the MM under local anesthesia on one hip, with direct comparison to standard needle serial marrow aspirates on the patients opposite hip (up to 350 ml per side). Cell viability, counts, CD34+, T cell, and MSC populations were assessed by flow cytometry. RESULTS: The MM successfully harvested marrow from a single entry sites and 2–3 paths under local anesthesia, without complications. Compared to standard harvest in the same patients, MM harvests had significantly number of Total Nucleated Cells ml compared to marrow harvested from the same patient by standard needle ( mean 1.98 fold greater TNC (range 0.87–3.36, p&lt;.05). Viability was equivalent at (&gt;99). In addition to higher TNC/ml, significantly higher levels (mean 3.56 fold) of Aldeflour/ALDH+ cells/ml, CD34+, and phenotypic MSC (CD45−,34−,90+,105+) and endothelial progenitor cells were obtained, as measured by flow cytometry. Mean CD3+ T-cell counts per ml were lower with MM harvests. CONCLUSIONS: The novel FDA approved MarrowMiner system demonstrated safety and efficacy in clinical use, harvesting more stem cells per unit volume in a single entry compared to standard harvest methods. These results suggests the MM may enable improved clinical stem cell harvests in a more rapid and minimally invasive manner in the outpatient setting, while harvesting a richer marrow product with less peripheral blood contamination. Such a system, facilitating convenient, on demand stem cell collection may have significant application for BMT and other marrow based cellular therapies.

Loss of P-Selectin Promotes the Function of Aged Hematopoietic and Leukemic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1577-1577
Author(s):  
Yaoyu Chen ◽  
Sullivan Con ◽  
Yiguo Hu ◽  
Linghong Kong ◽  
Cong Peng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Post Transplant ◽  
Marrow Cells

Abstract Abstract 1577 Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between the blood cells and their microenvironment. Adhesion molecules like P-selectin are essential to hematopoiesis, and their dysregulation has been implicated in leukemogenesis. We have previously shown a role for P-selectin in chronic myeloid leukemia and demonstrated that in its absence the disease process accelerates. Recently, there has also been speculation that P-selectin may play a role in the aging hematopoietic stem cells (HSCs), as its expression in upregulated as a mouse ages. In this study, we show that the loss of P-selectin function dysregulates the balance of stem cells and progenitors and that these differences become more pronounced with age. We compared the percentages of HSCs, long-term (LT)-HSCs, short-term (ST)-HSCs, multipotent progenitors (MPPs), CMPs, GMPs and MEPs in bone marrow by flow cytometry between wild type (WT) and Selp-/- mice. An age-dependent LT-HSC expansion was observed in WT mice. However, this expansion was prevented by the loss of Selp as observed in Selp-/-mice. Further, we demonstrate that with age LT-HSCs in particular express more elevated levels of P-selectin. LT-HSCs and ST-HSC/MPPs were isolated from the bone marrow of young (2 months old) and old (15 months old) WT mice and examined P-selectin expression by FACS. A significant increase in P-selectin expression was observed in LT-HSCs of old mice, and this increase was not observed in the ST-HSC+MPP subpopulations. We also show that the loss of P-selectin gene has profound effects of stem cell function, altering the capacity of these cells to home. Despite impaired homing capacity, stem cells lacking P-selectin possess a competitive advantage over their wild type counterparts. Using a stem cell competition assay, HSCs derived from Selp-/- mice (CD45.2+) and WT control mice (CD45.2+GFP+) were mixed in 1:1 ratio and transplanted into irradiated WT recipients (CD45.1). The initial findings were potentially indicative of the ability of cells derived from GFP mice to more efficiently home and engraft. Despite this initial advantage, cells derived from Selp-/- eventually exhibited a competitive and statistically significant advantage over the cells derived from GFP mice. At 30 days post-transplant, 49.9±1.4% of the CD45.2 subpopulation was GFP+. At 86 days post-transplant, 25.7±3.3 % of the CD45.2 cells derived from the peripheral blood were GFP+. Similarly, 23.0±3.7% of the CD45.2 cells derived from the bone marrow of these mice were GFP+. Indeed, we demonstrate that recipients of P-selectin deficient bone marrow cells more efficiently repopulate the bone marrow than controls and that this advantage extends and expands in the long-term. Finally, we demonstrate that recipients of leukemic cells lacking P-selectin develop a more accelerated form of leukemia accompanied by significant increases in stem and progenitor cells. Bone marrow cells from donor WT and Selp-/- mice were infected with retrovirus expressing BCR-ABL-GFP, and irradiated WT recipients were transplanted with 2×105 of these transduced donor cells. At 14 days post-transplant, recipient mice from each of the groups were sacrificed, and bone marrow cells were harvested and analyzed by flow cytometry. Recipients of leukemic Selp-/- cells possessed 3.5-fold more LSCs than recipients of wild-type cells. There were 3.1-fold more LT-LSCs and 3.8-fold more ST-LSCs and MPPs in recipients of Selp-/- cells than WT cells. In addition, recipients of leukemic Selp-/- cells possessed significantly more CMP (16.9-fold) and MEP (4.5-fold) cells. Because P-selectin expression increases with age on LT-HSCs, we sought to determine the role that age plays in CML development and progression. Bone marrow cells derived from 15-month-old donor Selp-/- and WT mice were transduced with BCR-ABL, respectively, followed by transplantation of the transduced cells into recipient mice. All recipients of BCR-ABL transduced Selp-/- cells died by 23 days after induction of CML and had a median survival of 19 days, whereas recipients of the transduced WT cells survived significantly longer. This pro-leukemic role for cells lacking P-selectin expression is leukemic stem cell-specific rather than stromal cell-specific and supports an essential role for P-selectin on leukemic stem cells. Disclosures: No relevant conflicts of interest to declare.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of bone marrow-derived stem cells, renal progenitor cells and stem cell factor in chronic renal allograft nephropathy

2013 ◽  
Vol 49 (3) ◽  
pp. 235-247
Author(s):  
Hayam Abdel Meguid El Aggan ◽  
Mona Abdel Kader Salem ◽  
Nahla Mohamed Gamal Farahat ◽  
Ahmad Fathy El-Koraie ◽  
Ghaly Abd Al-Rahim Mohammed Kotb
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Renal Allograft ◽  
Renal Progenitor Cells ◽  
Renal Progenitor

scholarly journals PTPN11D61Y/+; Vavcre+ Animals Commonly Succumb to Leukemia Relapse Despite Robust Engraftment of Transplanted Wild-Type Hematopoietic Stem and Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 496-496
Author(s):  
Stefan P. Tarnawsky ◽  
Mervin C. Yoder ◽  
Rebecca J. Chan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Stem And Progenitor Cells ◽  
Leukemia Relapse ◽  
Conditioning Regimens

Juvenile Myelomonocytic Leukemia (JMML) is a rare childhood myelodysplastic / myeloproliferative overlap disorder. JMML exhibits myeloid populations with mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. While allogeneic hematopoietic stem cell transplant (HSCT) is the treatment of choice for children with JMML, 50% of children succumb to leukemia relapse; however, the mechanism leading to this high relapse rate is unknown. We hypothesized that the hyperinflammatory nature of JMML may damage the bone marrow microenvironment, leading to poor engraftment of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and thus promoting leukemia relapse. Using Vav1 promoter-directed Cre, we generated a mouse model of JMML that conditionally expresses gain-of-function PTPN11D61Yin utero during development. While PTPN11D61Y/+; VavCre+embryos did not demonstrate in utero lethality, we observed a modest reduction of PTPN11D61Y/+; VavCre+ mice at the time of weaning compared to predicted Mendelian frequencies. Further, surviving PTPN11D61Y/+; VavCre+ mice developed elevated peripheral blood leukocytosis and monocytosis as early as 4 weeks of age compared to PTPN11+/+; VavCre+ controls. To address the hypothesis that an aberrant bone marrow microenvironment in the PTPN11D61Y/+ mice leads to poor engraftment of wild-type donor cells following transplant, we examined engraftment of wild-type hematopoietic stem and progenitor cells (HSPCs) in the PTPN11D61Y/+; VavCre+ mice and monitored animals for disease relapse. 16-24 week-old diseased PTPN11D61Y/+; VavCre+ and control PTPN11+/+; VavCre+ mice were lethally irradiated (11 Gy split dose) and transplanted with 5 x 105 CD45.1+ wild-type bone marrow low density mononuclear cells (LDMNCs), which simulates a limiting stem cell dose commonly available in a human HSCT setting. 6 weeks post-HSCT, PTPN11D61Y/+; VavCre+recipients demonstrated an unexpected elevated CD45.1+ donor cell contribution in peripheral blood compared to the control PTPN11+/+; VavCre+ recipients. However, despite superior engraftment in the PTPN11D61Y/+; VavCre+ recipients, these mice had a significantly shorter median survival post-HSCT due to a resurgence of recipient CD45.2-derived leukemic cells. We repeated the experiment using a high dose of CD45.1+ LDMNCs (10 x 106 cells) to determine if providing a saturating dose wild-type cells could prevent the relapse of recipient-derived leukemogenesis and normalize the survival of the PTPN11D61Y/+; VavCre+recipients. While this saturating dose of wild-type cells resulted in high peripheral blood chimerism in both the PTPN11D61Y/+; VavCre+ and PTPN11+/+; VavCre+ recipients, the PTPN11D61Y/+; VavCre+ animals nevertheless demonstrated significantly reduced overall survival. When we examined the cause of mortality in the HSCT-treated PTPN11D61Y/+; VavCre+mice, we found enlarged spleens, hypercellular bone marrow, and enlarged thymuses. Flow cytometry revealed that the majority of cells in the peripheral blood, bone marrow, and spleen were recipient-derived CD45.2+ CD4+ CD8+ T cells. To verify that the disease was neoplastic in origin, secondary transplants into CD45.1/.2 recipients were performed from two independent primary PTPN11D61Y/+; VavCre+and two independent primary PTPN11+/+; VavCre+ controls. Secondary recipients of bone marrow from PTPN11D61Y/+; VavCre+ animals rapidly succumbed to a CD45.2-derived T-cell acute lymphoid leukemia (T-ALL). Previous studies demonstrated that wild-type PTPN11 is needed to protect the integrity of the genome by regulating Polo-like kinase 1 (Plk1) during the mitosis of the cell cycle (Liu et al., PNAS, 2016). We now demonstrate that even when PTPN11 mutant animals are provided with saturating doses of wild-type HSCs, dysregulated residual recipient cells are able to produce relapsed disease. Collectively, these studies highlight the propensity of residual mutant PTPN11 cells to transform after being subjected to mutagenic agents that are commonly used for conditioning regimens prior to allogeneic HSCT. These findings suggest that modified pre-HSCT conditioning regimens bearing reduced mutagenicity while maintaining adequate cytoreductive efficacy may yield lower post-HSCT leukemia relapse in children with PTPN11mutations. Disclosures No relevant conflicts of interest to declare.

Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Shai Erlich ◽  
Silvia R.P. Miranda ◽  
Jan W.M. Visser ◽  
Arie Dagan ◽  
Shimon Gatt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Acid Sphingomyelinase ◽  
Hematopoietic Stem

Abstract The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

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