Ectopic Expression of the Extracellular Domain of Mpl Is Sufficient to Induce a Hematopoietic Population Crisis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2886-2886
Author(s):  
Daniel C. Wicke ◽  
Johann Meyer ◽  
Guntram Büsche ◽  
Hans Kreipe ◽  
Zhixiong Li ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Ectopic Expression ◽  
Extracellular Domain ◽  
Target Cells ◽  
Severe Thrombocytopenia ◽  
Hematopoietic Stem ◽  
Platelet Counts ◽  
Cell Counts ◽  
Population Crisis

Abstract The thrombopoietin receptor Mpl is required for regeneration of hematopoietic stem cells and governs megakaryocytic differentiation. Patients with inherited MPL deficiency suffer from severe thrombocytopenia which progresses to aplastic anemia, a disorder called congenital amegakaryocytic thrombocytopenia (CAMT). As a first step towards a potential gene therapy for MPL deficiency, we retrovirally expressed the receptor in a murine bone marrow transplantation model. An initial series of vectors used a strong enhancer-promoter derived from the spleen-focus forming virus (SFFV). Mice transplanted with hematopoietic cells modified by these constructs developed a profound yet transient elevation of multi-lineage hematopoiesis due to increased signaling of the Thpo receptor on target and non-target cells. Mice developed increased leukocyte, erythrocyte and platelet counts (2–3 times elevated) compared to GFP control animals. Histopathology revealed an elevated number of mature megakaryocytes with atypical features like numerous giant forms in the bone marrow and the spleen and atypical neoformation of bone in the spleen leading to the diagnosis of chronic myeloproliferative disorder (CMPD). A minority of mice (3/27) developed erythroleukemia with almost 100% CD71 and TER119 double positive cells as detected by flow cytometry. Histopathology presented infiltration of erythroblasts in all hematopoietic tissues like the BM, spleen and liver. Molecular analysis revealed retroviral vector insertions in sfpi1, fli1 and klf3 that seem to be the major driving force for the development of leukemia in these animals. Somewhat unexpectedly, in the majority of mice the CMPD converted into a progressive, potentially lethal pancytopenia. Animals had severely reduced blood cell counts with only 50% of leukocyte, 20% of erythrocyte and 10% of platelet counts compared to GFP control animals. This population crisis affected all major blood lineages and also involved co-existing unmodified hematopoiesis. Histopathology presented a dysmegakaryopoiesis with an increased number of atypical micro-megakaryocytes, histiocytes with erythrocytophagocytosis and atypical mast cell proliferation diagnosed as a myelodysplastic syndrome (MDS)-like disorder. In the bone marrow, pancytopenic mice had reduced cell numbers of the primitive cell fraction (LSK cells). To address the mechanism of pancytopenia, we expressed a dominant negative form of Mpl (dnMpl) consisting of the extracellular and transmembrane domain and lacking the intracellular signal transduction domain. Animals transplanted with dnMpl-modified cells failed to show the initial CMPD but developed the same pancytopenic, MDS-like end stage. A vector expressing Mpl under control of the PGK promoter or a fragment of the Mpl-promoter reduced or completely avoided the side effects (CMPD, MDS-like disorder) observed with vectors using stronger promoters. The induction of a hematopoietic population homeostasis thus depends upon Mpl expression levels, indicating the need for strictly regulated transgene expression in gene therapy for MPL deficiency. As ectopic expression of the extracellular domain is sufficient to cause MDS, sequestration of crucial niche factors like Thpo may contribute to the pathogenesis of this disorder. This study demonstrates that ectopic expression of a hematopoietic growth factor receptor may disturb organ homeostasis through interference with intra- and extracellular mechanisms of cell communication.

Bleeding Phenotype of Murine Bernard Soulier Syndrome Is Potentially Corrected by Non-Myeloablative Hematopoietic Stem Cell Transplantation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3340-3340
Author(s):  
Sachiko Kanaji ◽  
Scot A. Fahs ◽  
Jerry Ware ◽  
Robert R. Montgomery ◽  
Qizhen Shi
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Bleeding Time ◽  
Mononuclear Cells ◽  
Platelet Glycoprotein ◽  
Mouse Bone Marrow ◽  
Therapeutic Effects ◽  
Hematopoietic Stem ◽  
Platelet Counts ◽  
Bernard Soulier Syndrome

Abstract Abstract 3340 Bernard Soulier Syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP)Ib/IX complex. Platelet transfusion is the primary treatment for hemorrhage but is often limited because of the potential for provoking allo-immunization and refractoriness. We have previously shown that the macrothrombocytopenia and prolonged bleeding of murine BSS (GPIbαnull) can be corrected by myeloablative total body irradiation (TBI) and transplantation of hematopoietic stem cells (HSCs) transduced with a lentivirus that expresses hGPIbα under integrin αIIb promoter control (2bIbαLV). For application of this gene therapy approach to the treatment of human patients, it is important to minimize treatment-related adverse effects. In this study, we established a transgenic mouse that expresses hGPIbα at levels similar to the average levels observed in 2bIbαLV transduced HSC recipients that can be used as a consistent source of bone marrow HSCs (hGPIbαtg+). We undertook two approaches for evaluation of HSC transplantation using hGPIbαtg+ mouse bone marrow (BM) cells; 1) determine the percentage of hGPIbαtg+ HSCs necessary for therapeutic benefit and 2) determine if non-myeloablative strategies can achieve hGPIbα expression levels sufficient to correct the bleeding phenotype of GPIbαnullmice. In order to determine the percentage of HSCs required to achieve therapeutic effects, mixed BM chimeras were generated by reconstituting lethally irradiated GPIbαnull mice with variable mixtures (5% - 100%) of BM cells isolated from hGPIbαtg+ and GPIbαnull mice. Tail bleeding time assays performed after BM reconstitution demonstrated that 10% hGPIbαtg+ BM mononuclear cells mixed with 90% GPIbαnull BM mononuclear cells were sufficient to correct the tail bleeding time (n=5). Platelet analysis showed that the bleeding phenotype was rescued in recipients having higher than 40 × 103/μl of hGPIbαtg+ platelets, which corresponds to 6.5% of wild type mouse platelet counts (630 ± 57 × 103/μl, n = 8). These results suggest therapeutic potential for non-myeloablative conditioning regimens for the treatment of murine BSS. Thus we next tested the transplantation of hGPIbαtg+ mouse BM cells into GPIbαnull recipients conditioned with two non-myeloablative doses of busulfan (25 mg/kg on days -2 and -1). Analysis of platelets by flow cytometry showed that, as expected, the percentage of hGPIbα-positive platelets in busulfan-conditioned recipients was lower than those transplanted after lethal TBI conditioning (63.2% ±36.6%, n = 15 vs. 95.2% ±1.7%, n = 8). However, tail bleeding time assays showed that bleeding times of busulfan-conditioned recipients were corrected and not significantly different from lethally irradiated recipients (2.7 ± 3.1 minutes, n = 15 vs 1.9 ± 1.5 minutes, n = 8), while no untreated GPIbαnull mice (n=8) stopped bleeding within 10 minutes. Platelet counts were also significantly increased in busulfan-conditioned recipients compared to untreated GPIbαnull mice (381 ± 132 × 103/μl, n = 11 vs. 181 ± 35 × 103/μl, n = 11). Thus non-myeloablative doses of busulfan conditioning and transplantation of hGPIbαtg+mouse BM cells were shown to achieve relevant levels of engraftment in murine BSS with significantly corrected bleeding times and platelet counts. Antibody response to hGPIbα and immune-mediated thrombocytopenia was documented in 3 of 15 recipient mice, suggesting immunogenicity of hGPIbα transgene protein in GPIbαnullmice partially immunosuppressed with busulfan. However, immunotolerance was identified without treatment and antibody disappeared in all three mice 3 to 7 months after BM transplantation. In conclusion, non-myeloablative doses of busulfan conditioning may be a reasonable alternative to TBI for the BMT-based treatment of BSS and could be utilized in non-myeloablative autologous gene therapy in human BSS. Disclosures: No relevant conflicts of interest to declare.

Retroviral Ectopic Expression of a Signaling-Defective Thrombopoietin Receptor (Mpl) Induces a Systemic Loss of Hematopoietic Stem Cells in Mice,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4175-4175
Author(s):  
Sabine Wintterle ◽  
Dirk Heckl ◽  
Adrian Schwarzer ◽  
Guntram Buesche ◽  
Christopher Baum ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Aplastic Anemia ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Severe Thrombocytopenia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Gene Sets

Abstract Abstract 4175 MPL signaling, induced by the binding of Thrombopoietin (THPO), regulates megakaryopoiesis and platelet development, and is essential for hematopoietic stem cell (HSC) maintenance. Murine lineage negative, Sca-1 and c-kit positive bone marrow (BM) cells (LSK cells) and human CD34 positive cells expressing MPL have long term BM reconstitution capacities. In human patients, deficiency of MPL due to inactivating mutations in the MPL gene causes severe thrombocytopenia and progressive aplastic anemia, which is lethal if not treated by BM transplantation (BMT). This rare genetic disorder is termed congenital amegakaryocytic thrombocytopenia (CAMT). With the final aim of developing gene therapy to treat CAMT, we explored the effects of ectopic Mpl expression in wild-type C57Bl/6 mice. The enforced overexpression of Mpl induced a rapid expansion of all three hematological cell lineages, resembling chronic myeloproliferative disease (CMPD) 2 months after BMT (n=25 mice). Surprisingly, mice subsequently developed life threatening pancytopenia with normal Thpo levels despite low platelet counts (167+/−52 × 103/μl). The BM showed reduced LSK cell numbers (0.01+/−0.01 % versus controls 0.05+/−0.015%, p=0.03). Non-transduced, co-transplanted BM cells could not rescue the disturbed hematopoiesis. Based on these observations, we hypothesized that the ectopic overexpression of Mpl in hematopoietic cells induces a dominant-negative effect, dysregulating the Thpo-Mpl balance. To test our hypothesis, we ectopically expressed a signaling-defective truncated dominant-negative Mpl (dnMpl) receptor, lacking the complete intracellular domain, in a BMT model in wild-type C57Bl/6 mice (n=23). Lineage negative cells were transduced with gammaretroviral vectors expressing dnMpl from the SFFV promoter in the LTRs and transplanted into lethally irradiated mice. We observed long term engraftment with 57.6+/−1.9% dnMpl positive mononuclear cells in the peripheral blood (controls 76.7+/−7.8%) after 19 weeks and severe thrombocytopenia (174 +/− 32 x103/μl) in the dnMpl expressing animals (controls 951+/−233×103/μl). After 23 weeks, we detected 15–34% dnMpl positive BM cells (controls 15–99%). To address the question of whether the BM of dnMpl treated mice contained fewer HSCs compared to control transplanted mice, we analyzed the LSK cell number and saw a ∼4.6-fold reduction (0.02+/− 0.005% versus control mice 0.07+/−0.012%; p=0.0016). As Thpo/Mpl signaling is known to induce HSC quiescence, we speculated that the loss of these signals induces HSC cycling. Cell cycle analysis of the LSK cells of dnMpl treated mice showed a transition into G1/S/G2, indicating a loss of quiescent HSCs (G0 3.5 +/−1.6% vs controls 29.8 +/−10.5%). This effect was even more pronounced in CD34 negative LSK cells (5.4+/−4.0% vs. 44.5+/−8.5%). Secondary transplantation of dnMpl BM showed severely reduced repopulation capacity with 5 out of 6 secondary recipients dying due to graft failure. In selected animals we transplanted another batch of BM after 16 weeks without preconditioning of the mice. Long term repopulation of the second graft was seen in 71–79% of dnMpl treated mice (controls 0.2–1.1%) and BM cells further engrafted in secondary recipients. To address changes in HSC gene expression, we sorted LSK cells from dnMpl BM (pool of 4–5 dnMpl mice for each sample) and also separated dnMpl positive and negative cells. Our controls were LSK cells from control transplanted mice (one per sample). RNA was analyzed on an Affymetrix Mouse Genome 430 2.0 array in triplicates. Unsupervised clustering and principle component analysis revealed that dnMpl positive and negative samples clustered, suggesting a systemic effect on HSCs. We tested more than 4000 gene sets from the MySigDB Database (Broad Institute, Cambridge, MA) for enrichment in either of the phenotypes and found a profound downregulation of gene sets containing “stemness” genes in the LSK cells of dnMpl treated mice, irrespective of cellular dnMpl expression. In summary, we show that ectopic expression of dnMpl in a subset of BM cells disturbs Thpo/Mpl-signaling inducing thrombocytopenia and a systemic loss of HSCs. Besides underlining the strict requirement for regulated Mpl expression in gene therapy for CAMT, these data create new hypotheses for the pathogenesis of aplastic anemia and the development of non-cytotoxic conditioning regimens in BMT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fully Closed, Large-Scale, and Clinical Grade Cell Sorting of Hematopoietic Stem Cell (HSC)-Enriched CD90+ Cells for Transplantation and Gene Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3246-3246
Author(s):  
Stefan Radtke ◽  
Margaret Cui ◽  
Anai M Perez ◽  
Yan-Yi Chan ◽  
Stefanie Schmuck ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Gene Modification ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Fractions

Introduction: Hematopoietic stem cell (HSC) gene therapy/editing is a viable treatment option for various hematological diseases and disorders including hemoglobinopathies and HIV/AIDS. Most if not all currently available approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly committed progenitor cells and only very few true HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy/editing approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side-effects in non-target cells. We recently described a novel HSC-enriched CD34 subset (CD90+CD45RA-) that is exclusively responsible for rapid recovery onset, robust long-term multilineage engraftment, as well as entire reconstitution of the bone marrow stem cell compartment in the nonhuman primate (NHP) stem cell transplantation and gene therapy model (Radtke et al. 2017, STM). Most importantly, we demonstrate that this CD34 subset reduces the number of target cells, modifying reagents and costs by more than 10-fold without compromising the long-term efficiency of gene-modification in the NHP (Humbert and Radtke et al. 2019, STM). Here, we aimed to develop a clinical protocol to reliably purify and efficiently gene-modify human HSC-enriched CD90+ cell fractions. Methods: Large-scale enrichment of CD34+ cells from GCSF-mobilized leukapheresis products was initially performed on the Miltenyi CliniMACS Prodigy according to previously established protocols (Adair et al. 2017, Nat. Comm.). Yield, purity, quality, and feasibility of CD90 sorting was then comprehensively tested on two different commercially available cell sorting systems comparing the jet-in-air sorter FX500 from Sony and the cartridge-based closed-system sorter MACSQuant Tyto from Miltenyi Biotech with our clinically approved gold-standard CD34-mediated gene therapy approach. Sorted CD90+ and bulk CD34+ cells were transduced with a clinical-grade lentivirus encoding for GFP and the multilineage differentiation as well as engraftment potential tested using in vitro assays and the NSG mouse xenograft model, respectively. Results: Flow-cytometric sort-purification of CD90+ cells was similarly efficient in purity and yield using either the FX500 or Tyto (Figure A,B). Both approaches reliably reduced the overall target cell count by 10 to 15-fold without impacting the cells viability and in vitro colony-forming cell potential. Unexpectedly, the transduction efficiency of sort-purified CD90+ cells was significantly improved compared to bulk-transduced CD34+ cells and especially the CD34+CD90+ subset (Figure C). All cell fractions demonstrated robust mouse xenograft potential (Figure D). Most importantly, significantly higher levels of GFP+ expression in the peripheral blood, bone marrow, spleen and thymus were observed after transplantation of gene-modified CD90+ compared to bulk CD34+ cells in NSG mice (Figure E). Conclusion: Here, we show that sort-purification of our HSC-enriched CD34+CD90+ cell subset is technically feasible and highly reproducible in two different systems. Purification of human CD90+ cell fractions significantly increased the gene-modification efficiency of primitive human HSCs with multilineage mouse engraftment potential. These findings should have important implications for currently available as well as future HSC gene therapy and gene editing protocols. Isolation of an HSC-enriched phenotype will allow more targeted gene modification and thus likely reduce unwanted off target effects. Our approach further reduced the overall costs for gene modifying reagents, can be combined with a closed transduction system, increase the portability and ultimately make HSC gene therapy GMP-facility independent and affordable. Finally, this stem cell selection strategy may also allow efficient and effective depletion of donor T cells in the setting of allogeneic stem cell or organ transplantation. Figure: A) Purity and B) yield of CD90+ cells after sort-purification. C) Transduction efficiency of bulk-transduced CD34+CD90+ cells and sort-purified CD90+ cells. Frequency of D) human chimerism and E) GFP+ human CD45+ cells in the peripheral blood (PB), bone marrow, spleen and thymus after transplantation of gene-modified bulk CD34+ or sort-purified CD90+ cells. Figure Disclosures Kiem: CSL Behring: Consultancy; Rocket Pharma: Consultancy, Equity Ownership; Homology Medicines: Consultancy, Equity Ownership; Magenta Therapeutics: Consultancy.

scholarly journals Understanding the Mechanisms Underlying Thrombocytopenia in Visceral Leishmaniasis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2378-2378 ◽  
Author(s):  
Gulab Fatima Rani ◽  
Olivier Preham ◽  
Ian Hitchcock ◽  
Paul Kaye
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Visceral Leishmaniasis ◽  
Conflicts Of Interest ◽  
Experimental Models ◽  
Severe Thrombocytopenia ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Platelet Counts ◽  
Splenic Macrophages

Visceral leishmaniasis (VL) is a neglected tropical parasitic disease caused by Leishmania parasites and only second to malaria in terms of worldwide morbidity and mortality. According to recent WHO report, there are 500,000 cases of VL worldwide leading to ~30,000 deaths per year. VL is endemic in 98 countries but the major disease burden is contributed by Brazil, India and Sudan. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulations and extensive hematological complications. We have shown previously using experimental models of infection that the infiltration of CD4+ T cells results in disruption to the bone marrow environment, resulting in dysfunctional hematopoietic stem and progenitor cells self-renewal (Pinto et al, PLOS Pathogens, 2017) and aberrant medullary erythropoiesis causing pathological anemia (Preham et al, Frontiers in Immunology, 2018). Thrombocytopenia is also dominant hematological feature seen in both human and experimental models that may reflect either reduced platelet production or enhanced clearance. However, the mechanisms of VL-driven thrombocytopenia remain poorly understood. The aim of this study is to explore the possible underlying mechanisms from platelet production to phagocytic cells dependent clearance. Using a murine experimental model of VL, we demonstrate a steady decrease in the platelet count from d14 onwards in infected mice culminating in severe thrombocytopenia on d28 of infection (infected: 225.9 ±35.7 vs naïve: 1005 ±90.6, x 106/µl). Critically, thrombocytopenia is completely reversible after a single dose of liposomal amphotericin B (Ambisome @ 8mg/kg bodyweight, IV) which clears parasites by delivering the drug directly to parasite harbouring tissue macrophages, thereby improving parasite clearance and reducing toxicity. Despite significant thrombocytopenia, the number and gross morphology of bone marrow megakaryocytes (MKs) were not altered, but MK ultrastructure studies using transmission electron microscopy identified significantly reduced demarcation membranes in infected mice compared to naïve. Levels of plasma thrombopoietin (TPO), the key regulator of MK differentiation and platelet production, were decreased in infected vs naïve mice (1254 ± 95.49 vs 3249 ± 125.1 pg/ml) and administration of exogenous TPO resulted in complete recovery of platelet counts. Given that the majority of TPO is produced by the liver, reduction in the levels of circulating TPO during infection is likely due to destruction of liver architecture by parasite loaded hepatic granulomas. Together, these data suggest that despite some changes in MK cytoplasmic maturation, the bone marrow microenvironment remains supportive of MK differentiation capacity during VL. As platelet production is not significantly altered by VL, we next determined effects on platelet clearance. Large number of highly active splenic macrophages are common in VL and are known for their phagocytic properties. Experiments conducted on VL-infected splenectomised mice demonstrated a reduction in thrombocytopenia compared to sham-operated infected mice (685 ±32 vs 297± 16, x 106/µl) and showed a great response to exogenous TPO, implying splenic clearance may be involved in thrombocytopenia. Partial depletion of splenic macrophages in infected mice using clodronate liposomes did not alter platelet count, whereas neutrophil deletion (anti-Gr1 mAb @ 250ug/g IP) in infected mice resulted in a near 2-fold increase in platelet counts. Furthermore, circulating platelets in VL infected mice were IgG coated compared to naive which is likely to further enhance autoimmune platelet clearance. Severe thrombocytopenia and bleeding are important clinical manifestations of VL. Our findings clearly demonstrate that the mechanisms of thrombocytopenia in VL are multifactorial but do not cause permanent long term damage to the BM microenvironment. Critically, these changes could be reversed rapidly by clearing parasitemia, using TPO agonists to increase numbers of circulating platelets and/or by reducing platelet clearance. This highlights the possibility of re-evaluating the current treatment regimen in VL endemic countries by including therapeutic interventions aimed at reversing severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

STAT3 Deletion In Hematopoietic Tissue Causes Mitochondrial Dysfunction with Increased ROS Leading to LT-Repopulating Stem Cell Depletion, Erythroid Dysplasia, Peripheral Neutrophilia, and a Rapid Aging-Like Phenotype.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2621-2621
Author(s):  
Charlie Mantel ◽  
Steve Messina-Graham ◽  
Akira Moh ◽  
Xin-Yuan Fu ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mitochondrial Function ◽  
Myeloid Cell ◽  
Cell Depletion ◽  
Target Cells ◽  
Hematopoietic Tissue ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
First Time

Abstract Abstract 2621 The essential transcription factor, Stat3, has been extensively studied in the context of cytokine signal transduction. Tissue-specific targeted deletion of STAT3 has been studied in heart, liver, and other tissues. In mouse hematopoietic tissue, STAT3 deletion (STAT3-KO mice) results in uniform post-natal death about 6–8 weeks after birth, although we show here that the pups are born with normal peripheral lymphoid and myeloid blood cell counts and pups appear normal until 4–5 weeks old. Then an aging-like shift in the lymphoid/myeloid ratio quickly occurs and the animals die within about 2 weeks of this shift. These animals suffer from intestinal inflammation, leukocyte infiltration in various tissues/organs, osteoporosis, dysfunctional innate immunity, myeloid cell overproduction and dendritic cell depletion. These findings are believed to be due, primarily, to abnormal cytokine signaling, however, the exact cause of death remains unknown. Stat3 has recently been implicated as a positive regulator of mitochondrial respiration and function. Mitochondrial function has been linked to aging and aging-related diseases like myelodysplastic syndromes (MDS), anemia, and erythroid dysplasia. It is therefore possible that Stat3 deletion in hematopoietic tissue results in disruption of mitochondrial regulation, which could also contribute to the observed phenotype of STAT3-KO mice. We have now investigated the function of mitochondria in the bone marrow stem/progenitor compartment of these mice as well as hematopoietic stem cell (HSC) repopulating ability and other cytokine responses. We show, for the first time, that STAT3 deletion causes pronounced erythroid dysplasia and anemia with erythrocyte hypochromasia and codocytes (target cells), aging-like lymphoid/myeloid ratio shift, hypersegmented neutrophilia, pronounced bone marrow accumulation of immature myeloid cells (bands), erythroblast depletion, and splenic hypertrophy and enlargement that appears to be due to erythroid and myeloid cell infiltration suggesting a shift in the primary site of erythropoiesis from the bone marrow to the spleen. Colony formation from bone marrow myeloid progenitors was unaffected when stimulated with single cytokines, but synergistic cytokine combinations failed to demonstrate proliferative synergy. HSC competitive repopulating ability was significantly diminished, especially in female STAT3 KO donors, but secondary transplants show that HSC self-renewal potential may not be affected, thus suggesting a reduced HSC number/function in STAT3 KO mouse bone marrow. This is consistent with the finding of significantly decreased numbers of CD34−SLAM+LSK cells. Mitochondrial mass was significantly increased in CD34+LSK subpopulation, but mitochondrial function (membrane potential, ΔΨm) was significantly diminished in STAT3 KO mouse LSK cells, which is consistent with the proposed positive role of Stat3 in mitochondrial function and also consistent with the finding of significantly increased ROS levels in bone marrow LSK subpopulations. Graded loss of Sca-1 surface marker (differentiation) in these cells was associated a with a graded increase in ΔΨm (activation); STAT3 KO mouse LSK cells displayed premature mitochondrial activation during differentiation to Sca-1− progenitors. These data show, for the first time, that in-vivo deletion of STAT3 in mouse hematopoietic stem/progenitor cells results in defective mitochondrial function with increased ROS production along with disrupted differentiation-driven mitochondrial biogenesis and activation in these cells. This likely contributes to an aging-like phenotype of the hematopoietic system with lymphoid/myeloid shift, anemia, non-sideroblastic erythroid dysplasia, myeloproliferative dysfunction, and diminished HSC number/function and raises the possibility that aberrant Stat3/mitochondria regulation could contribute to aging-associated diseases like MDS and progressive anemias. Disclosures: Broxmeyer: Corduse: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Immature platelet values indicate impaired megakaryopoietic activity in neonatal early-onset thrombocytopenia

2010 ◽  
Vol 103 (05) ◽  
pp. 1016-1021 ◽  
Author(s):  
Hannes Hammer ◽  
Christoph Bührer ◽  
Christof Dame ◽  
Malte Cremer ◽  
Andreas Weimann
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Intensive Care ◽  
Early Onset ◽  
Absolute Number ◽  
Severe Thrombocytopenia ◽  
Platelet Counts ◽  
Immature Platelet Fraction ◽  
Neonatal Thrombocytopenia ◽  
Immature Platelets

SummaryNewly released platelets, referred to as immature platelets, can be reliably quantified based on their RNA content by flow cytometry in an automated blood analyser. The absolute number of immature platelets (IPF#) and the immature platelet fraction (IPF%) reflect megakaryopoietic activity. We aimed to analyse the implication of these parameters in analysing the pathomechanism of early-onset neonatal thrombocytopenia. Platelet counts and IPF were determined at day 1 to 3 (d1 to d3) in 857 neonates admitted to intensive care. In thrombocytopenic patients (platelet counts<150 x 109/l, n=129), IPF# was significantly lower (8.5 ± 2.7 x 109/l), than in non-thrombocytopenic neonates (9.5 ± 3.6 x 109/l, n=682, p<0.05). IPF% was significantly higher in thrombocytopenic (9.3 ± 7.9%) vs. non-thrombocytopenic neonates (4.1 ± 1.8%, p<0.001). While neonates with early-onset infection (n=134) had lower platelet counts (199 ± 75 x 109/l) compared to controls (230 ± 68 x 109/l, n=574, p<0.01), there were no differences in IPF# or IPF%. Likewise, “small for gestational age” infants (SGA, n=149) had lower platelet counts at d1 (199 ± 75 x 109/l, p<0.001) than controls, but no differences in IPF. A trend towards lower IPF# was detected if SGA infants with platelet counts <100 x 109/l (5.4 ± 3.9 x 109/l, n=11) and thrombocytopenic neonates with infection (9.9 ± 7.3 x 109/l, n=10, p=0.11) were compared. The evaluation of IPF# indicates that thrombocytopenia in neonates is likely due to a combination of increased platelet consumption and inadequate megakaryopoietic response by the neonatal bone marrow. Furthermore, SGA neonates with moderate and severe thrombocytopenia might have a pronounced suppression of megakaryopoiesis compared to neonates with infection.

Hematologic Toxicity of High-Dose Iodine-131–Metaiodobenzylguanidine Therapy for Advanced Neuroblastoma

2004 ◽  
Vol 22 (12) ◽  
pp. 2452-2460 ◽  
Author(s):  
Steven G. DuBois ◽  
Julia Messina ◽  
John M. Maris ◽  
John Huberty ◽  
David V. Glidden ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Radiation Dose ◽  
Red Cells ◽  
Whole Body ◽  
High Dose ◽  
Hematologic Toxicity ◽  
Severe Thrombocytopenia ◽  
Hematopoietic Stem ◽  
Iodine 131 ◽  
Mibg Therapy

Purpose Iodine-131–metaiodobenzylguanidine (131I-MIBG) has been shown to be active against refractory neuroblastoma. The primary toxicity of 131I-MIBG is myelosuppression, which might necessitate autologous hematopoietic stem-cell transplantation (AHSCT). The goal of this study was to determine risk factors for myelosuppression and the need for AHSCT after 131I-MIBG treatment. Patients and Methods Fifty-three patients with refractory or relapsed neuroblastoma were treated with 18 mCi/kg 131I-MIBG on a phase I/II protocol. The median whole-body radiation dose was 2.92 Gy. Results Almost all patients required at least one platelet (96%) or red cell (91%) transfusion and most patients (79%) developed neutropenia (< 0.5 × 103/μL). Patients reached platelet nadir earlier than neutrophil nadir (P < .0001). Earlier platelet nadir correlated with bone marrow tumor, more extensive bone involvement, higher whole-body radiation dose, and longer time from diagnosis to 131I-MIBG therapy (P ≤ .04). In patients who did not require AHSCT, bone marrow disease predicted longer periods of neutropenia and platelet transfusion dependence (P ≤ .03). Nineteen patients (36%) received AHSCT for prolonged myelosuppression. Of patients who received AHSCT, 100% recovered neutrophils, 73% recovered red cells, and 60% recovered platelets. Failure to recover red cells or platelets correlated with higher whole-body radiation dose (P ≤ .04). Conclusion These results demonstrate the substantial hematotoxicity associated with high-dose 131I-MIBG therapy, with severe thrombocytopenia an early and nearly universal finding. Bone marrow tumor at time of treatment was the most useful predictor of hematotoxicity, whereas whole-body radiation dose was the most useful predictor of failure to recover platelets after AHSCT.

scholarly journals Effects of recombinant human interleukin-11 on hematopoietic reconstitution in transplant mice: acceleration of recovery of peripheral blood neutrophils and platelets

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 27-34 ◽  
Author(s):  
XX Du ◽  
T Neben ◽  
S Goldman ◽  
DA Williams
Keyword(s):  
Peripheral Blood ◽  
Daily Administration ◽  
Total Leukocyte Count ◽  
Mouse Bone Marrow ◽  
Blood Leukocytes ◽  
Hematopoietic Stem ◽  
Platelet Counts ◽  
Cell Counts ◽  
Interleukin 11

Abstract We have examined the effects of recombinant human interleukin-11 (rhIL- 11) on the recovery of peripheral blood cell counts and proliferation of progenitors and hematopoietic stem cells (day 12 colony-forming units-spleen-CFU-S12) in vivo using a mouse bone marrow (BM) and spleen cell transplantation model. Recovery of leukocytes was accelerated in animals receiving daily administration of rhIL-11 (100 micrograms/kg/d) and reached normal levels by day 14 posttransplantation. This increased total leukocyte count reflected mainly an increase in neutrophils. Neutropenia (absolute neutrophil count [ANC] < 1,500) was present in control transplant mice for 14 to 15 days, while in the rhIL-11-treated group, neutrophils recovered to normal by days 8 to 10 and continued to increase until day 19. Animals treated with rhIL-11 had only 1 day with ANC demonstrated < 500. Correspondingly, rhIL-11 treatment increased granulocyte-macrophage progenitors (CFU-GM) derived from both spleen and BM cells. Higher doses of IL-11 increased CFU-GM nearly threefold and CFU-Mix fourfold to fivefold, while increasing burst-forming units- erythroid to a lesser degree. BM and spleen cellularity were both increased in IL-11-treated mice, but no increase in CFU-S12 was noted. In addition, in vivo daily administration of IL-11 increased peripheral platelet counts by threefold over control transplant mice at day 10 posttransplantation during the post-irradiation platelet nadir. Further treatment led to platelet counts higher than normal 18 days posttransplantation when control animals had just attained normal platelet counts. IL-11 can accelerate the recovery of the peripheral blood leukocytes, mainly neutrophils, and platelets in transplant mice, effects that may be clinically useful in future applications for BM transplantation and chemotherapy-related cytopenias.

scholarly journals Gpr44 Mediates the Selenium-Dependent Anti-Leukemic Effect in Acute Myeloid Leukemia

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1835-1835
Author(s):  
Fenghua Qian ◽  
Fenghua Qian ◽  
Diwakar Tukaramrao ◽  
Jiayan Zhou ◽  
Nicole Palmiero ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Murine Model ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Pparγ Agonist ◽  
Acute Myeloid

Abstract Objectives The relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia stem cells (LSCs) that are not targeted by existing therapies. LSCs show sensitivity to endogenous cyclopentenone prostaglandin J (CyPG) metabolites that are increased by dietary trace element selenium (Se), which is significantly decreased in AML patients. We investigated the anti-leukemic effect of Se supplementation in AML via mechanisms involving the activation of the membrane-bound G-protein coupled receptor 44 (Gpr44) and the intracellular receptor, peroxisome proliferator-activated receptor gamma (PPARγ), by endogenous CyPGs. Methods A murine model of AML generated by transplantation of hematopoietic stem cells (HSCs- WT or Gpr44−/−) expressing human MLL-AF9 fusion oncoprotein, in the following experiments: To investigate the effect of Se supplementation on the outcome of AML, donor mice were maintained on either Se-adequate (Se-A; 0.08–0.1 ppm Se) or Se-supplemented (Se-S; 0.4 ppm Se) diets. Complete cell counts in peripheral blood were analyzed by hemavet. LSCs in bone marrow and spleen were analyzed by flow cytometry. To determine the role of Gpr44 activation in AML, mice were treated with Gpr44 agonists, CyPGs. LSCs in bone marrow and spleen were analyzed. Mice transplanted with Gpr44−/- AML cells were compared with mice transplanted with wild type AML cells and the progression of the disease was followed as above. To determine the role of PPARγ activation in AML, PPARγ agonist (Rosiglitazone, 6 mg/kg, i.p, 14 d) and antagonist (GW9662, 1 mg/kg, i.p. once every other day, 7 injections) were applied to Se-S mice transplanted with Gpr44−/- AML cells and disease progression was followed. Results Se supplementation at supraphysiological levels alleviated the disease via the elimination of LSCs in a murine model of AML. CyPGs induced by Se supplementation mediate the apoptosis in LSCs via the activation of Gpr44 and PPARγ. Conclusions Endogenous CyPGs produced upon supplementation with Se at supraphysiological levels improved the outcome of AML by targeting LSCs to apoptosis via the activation of two receptors, Gpr44 and PPARg. Funding Sources NIH DK 07,7152; CA 175,576; CA 162,665. Office of Dietary Supplements, USDA Hatch funds PEN04605, Accession # 1,010,021 (KSP, RFP).

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3216-3226 ◽  
Author(s):  
Aisha V. Sauer ◽  
Emanuela Mrak ◽  
Raisa Jofra Hernandez ◽  
Elena Zacchi ◽  
Francesco Cavani ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Severe Combined Immunodeficiency ◽  
Intrinsic Defect ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Enzyme Replacement ◽  
Bone Phenotype

Abstract Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

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