scholarly journals Thalassemia Gene Therapy By In Vivo Transduction of Mobilized Hematopoietic Stem Cells (HSCs) with an Integrating Hybrid Adenovirus Vector System

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2193-2193
Author(s):  
Afrodite Georgakopoulou ◽  
Hongjie Wang ◽  
Chrysi Kapsali ◽  
Nikoleta Psatha ◽  
Angeliki Koufali ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematological Parameters ◽  
Sleeping Beauty ◽  
Vector System ◽  
Good Tolerability ◽  
Hematopoietic Stem

Abstract To overcome the cost and complexity of current thalassemia ex vivogene therapy protocols, we developed a minimally invasive and readily translatable approach for in vivo HSC gene delivery which abrogates the need for HSC leukapheresis, CD34+ cell selection, ex vivo HSC culture, myeloablation and ultimately, transplantation. Our approach involves HSC mobilization with G-CSF/AMD3100 andintravenous injections of a hybrid vector system consisting ofa CD46-targeting, helper-dependent adenoviral vector and the hyperactive Sleeping Beauty transposase (SB100x) that mediates integration of thevector-encoded γ-globin and mgmtP140K genes. Pretreatment with glucocorticoids before virus injectionsis used to blockthe release of pro-inflammatory cytokines andimmunosuppression is applied in order to avoid responses against human g-globin- and MGMT protein-expressing cells. We tested our approach in a mouse model recapitulating the phenotypeof human β-thalassemia intermedia (Hbbth-3/hCD46++ mice). At week 8 post transduction, hCD46+/+/Hbbth-3 mice expressed HbF in 31.2±2.7% of circulating erythrocytes. Due to a significant drop in HbF expression by week 16 (11.9±3.0%), a 4-dose O6BG/BCNU treatment was administeredin order to in vivo select forgene corrected hematopoietic progenitors, thus recovering the HbF expression in76.0±5.7% of the circulating erythrocytes, by week 29 post in vivo transduction. With an average vector copy number of 1.4/cell, the human γ-globin to mouse α-globin expression was ~10% by HPLC and the human γ-globin to mouse β-globin mRNA ratio ~10%, by qRT-PCR. Hematological parameters (RBCs, Ht, Hb, MCV, RDW, Reticulocytes) at week 29 post in vivo transduction, were significantly improved over baseline or were indistinguishable from normal values, suggesting near to complete phenotypic correction. Treated mice showed significant reduction of spleen size, extramedullary erythropoiesis and parenchymal hemosiderosis. After secondary transplantation and without in vivo selection, more than 90% of donor-derived erythrocytes (CD46+) were g-globin-positive, up to 20 weeks post-transplant. Safety was demonstrated by the good tolerability of treatment, the absence of alterations in hematopoiesis, the normal colony-forming potential of bone marrow cells and the random integration pattern of our vector system. Overall, we present a simplified platform for gene therapy of thalassemia, which can serve as a cost-efficient and "portable" approach to make gene therapy accessible even to resource-poor regions where thalassemia major is endemic but only minimally complex strategies could be adopted. Disclosures No relevant conflicts of interest to declare.

scholarly journals In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors

Blood ◽  
2016 ◽  
Vol 128 (18) ◽  
pp. 2206-2217 ◽  
Author(s):  
Maximilian Richter ◽  
Kamola Saydaminova ◽  
Roma Yumul ◽  
Rohini Krishnan ◽  
Jing Liu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Genetically Modify ◽  
Blood Stream ◽  
Vector System ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice

Abstract Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35++) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34+ cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking in BM HSPCs (Lin−Sca1+Kit− cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Abstract Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5143-5143
Author(s):  
Liesbeth De Waele ◽  
Kathleen Freson ◽  
Chantal Thys ◽  
Christel Van Geet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Phosphoglycerate Kinase ◽  
Lentiviral Vectors ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

Gene Therapy Corrects the Lethal Bone Marrow Failure in a Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 513-513
Author(s):  
Pekka Jaako ◽  
Shubhranshu Debnath ◽  
Karin Olsson ◽  
Axel Schambach ◽  
Christopher Baum ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Stem And Progenitor Cells

Abstract Abstract 513 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical abnormalities and predisposition to cancer. Mutations in genes that encode ribosomal proteins have been identified in approximately 60–70 % of the patients. Among these genes, ribosomal protein S19 (RPS19) is the most common DBA gene (25 % of the cases). Current DBA therapies involve risks for serious side effects and a high proportion of deaths are treatment-related underscoring the need for novel therapies. We have previously demonstrated that enforced expression of RPS19 improves the proliferation, erythroid colony-forming potential and differentiation of patient derived RPS19-deficient hematopoietic progenitor cells in vitro (Hamaguchi, Blood 2002; Hamaguchi, Mol Ther 2003). Furthermore, RPS19 overexpression enhances the engraftment and erythroid differentiation of patient-derived hematopoietic stem and progenitor cells when transplanted into immunocompromised mice (Flygare, Exp Hematol 2008). Collectively these studies suggest the feasibility of gene therapy in the treatment of RPS19-deficient DBA. In the current project we have assessed the therapeutic efficacy of gene therapy using a mouse model for RPS19-deficient DBA (Jaako, Blood 2011; Jaako, Blood 2012). This model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of Collagen A1 gene. Transgenic animals were bred either heterozygous or homozygous for the shRNA-D in order to generate two models with intermediate or severe Rps19 deficiency, respectively. Indeed, following transplantation, the administration of doxycycline to the recipients with homozygous shRNA-D bone marrow results in an acute and lethal bone marrow failure, while the heterozygous shRNA-D recipients develop a mild and chronic phenotype. We employed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the SFFV promoter, followed by IRES and GFP (SFFV-RPS19). A similar vector without the RPS19 cDNA was used as a control (SFFV-GFP). To assess the therapeutic potential of the SFFV-RPS19 vector in vivo, transduced c-Kit enriched bone marrow cells from control and homozygous shRNA-D mice were injected into lethally irradiated wild-type mice. Based on the percentage of GFP-positive cells, transduction efficiencies varied between 40 % and 60 %. Three months after transplantation, recipient mice were administered doxycycline in order to induce Rps19 deficiency. After two weeks of doxycycline administration, the recipients transplanted with SFFV-RPS19 or SFFV-GFP control cells showed no differences in blood cellularity. Remarkably, at the same time-point the recipients with SFFV-GFP homozygous shRNA-D bone marrow showed a dramatic decrease in blood cellularity that led to death, while the recipients with SFFV-RPS19 shRNA-D bone marrow showed nearly normal blood cellularity. These results demonstrate the potential of enforced expression of RPS19 to reverse the severe anemia and bone marrow failure in DBA. To assess the reconstitution advantage of transduced hematopoietic stem and progenitor cells with time, we performed similar experiments with heterozygous shRNA-D bone marrow cells. We monitored the percentage of GFP-positive myeloid cells in the peripheral blood, which provides a dynamic read-out for bone marrow activity. After four months of doxycycline administration, the mean percentage of GFP-positive cells in the recipients with SFFV-RPS19 heterozygous shRNA-D bone marrow increased to 97 %, while no similar advantage was observed in the recipients with SFFV-RPS19 or SFFV-GFP control bone marrow, or SFFV-GFP heterozygous shRNA-D bone marrow. Consistently, SFFV-RPS19 conferred a reconstitution advantage over the non-transduced cells in the bone marrow. Furthermore, SFFV-RPS19 reversed the hypocellular bone marrow observed in the SFFV-GFP heterozygous shRNA-D recipients. Taken together, using mouse models for RPS19-deficient DBA, we demonstrate that the enforced expression of RPS19 rescues the lethal bone marrow failure and confers a strong reconstitution advantage in vivo. These results provide a proof-of-principle for gene therapy in the treatment of RPS19-deficient DBA. Disclosures: No relevant conflicts of interest to declare.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

scholarly journals Pre-Existing Anti-FVIII Immunity Alters Therapeutic Platelet-Targeted FVIII Engraftment in the System Preconditioned with Busulfan Alone through Cytotoxic CD8 T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 444-444
Author(s):  
Weiqing Jing ◽  
Christina Baumgartner ◽  
Feng Xue ◽  
Jocelyn A. Schroeder ◽  
Qizhen Shi
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Cd8 T Cells ◽  
Hemophilia A ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Specific Response ◽  
Hematopoietic Stem ◽  
Fviii Inhibitor

Abstract The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model. FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period. We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway. In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 20-20
Author(s):  
James Bartram ◽  
Baobao (Annie) Song ◽  
Juying Xu ◽  
Nathan Salomonis ◽  
H. Leighton Grimes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Functional Decline ◽  
Functional Heterogeneity ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Marrow Cells

Abstract Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. Disclosures No relevant conflicts of interest to declare.

scholarly journals Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Blood ◽  
2018 ◽  
Vol 131 (26) ◽  
pp. 2915-2928 ◽  
Author(s):  
Chang Li ◽  
Nikoletta Psatha ◽  
Pavel Sova ◽  
Sucheol Gil ◽  
Hongjie Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Binding Site ◽  
Genome Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Cell Genome ◽  
Hsc Transplantation

Key Points CRISPR/Cas9-mediated disruption of a BCL11A binding site in HSCs of β-YAC mice results in the reactivation of γ-globin in erythrocytes. Our approach for in vivo HSC genome editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.

Oxidative Stress-Mediated Activation of AKT/mTOR Signaling Pathway Leads to Myeloproliferative Syndrome in FoxO3 Null Mice: A Role for Lnk Adaptor Protein

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 509-509 ◽  
Author(s):  
Safak Yalcin ◽  
Sathish Kumar Mungamuri ◽  
Dragan Marinkovic ◽  
Xin Zhang ◽  
Wei Tong ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Reactive oxygen species (ROS) are toxic byproducts of oxidative metabolism implicated in many debilitating human disorders including hematological malignancies and aging. ROS are also generated by growth factors and cytokine stimulation and play critical functions in normal cellular signaling. However, not much is known of how ROS impact physiological processes in normal and diseased states. We and others have recently shown critical functions for box (O) family of forkhead transcription factors (Fox)O in the regulation of physiological ROS in primitive hematopoietic cells. In particular, FoxO3 has emerged as the principal FoxO whose regulation of ROS is essential for the maintenance of hematopoietic stem cell pool. Although FoxO3’s activity is constitutively repressed by several oncoproteins that play critical roles in myeloproliferative disorders the role of FoxO3 in the regulation of primitive hematopoietic progenitors remains elusive. FoxO’s function is restrained by AKT serine threonine protein kinase. AKT supports growth, survival and proliferation by promoting inhibition of FoxO and activation of the mammalian target of rapamycin (mTOR) and its downstream target p70 S6 Kinase (S6K) through phosphorylation. We demonstrate that loss of FoxO3 leads to a myeloproliferative-like syndrome characterized by leukocytosis, splenomegaly, enhanced generation of primitive progenitors including colony-forming-unit-spleen (CFU-S) in hematopoietic organs and hypersensitivity of hematopoietic progenitor cells to cytokines in FoxO3 null mice. These findings were intriguing since we had not found FoxO3 null hematopoietic stem cells to exhibit enhanced cycling in vivo or to generate excessive hematopoietic progenitors ex vivo (Yalcin et al., JBC, 2008). To investigate the mechanism of enhanced myeloproliferation, we interrogated cytokine-mediated activation of signaling pathways in freshly isolated FoxO3 null versus wild type bone marrow cells enriched for hematopoietic progenitors. To our surprise we found that stimulation with cytokines including IL-3 led to hyperphosphorylation of AKT, mTOR and S6K but not STAT5 proteins in FoxO3 null as compared to wild type cytokine-starved hematopoietic progenitors. In agreement with these results, in vivo administration of the mTOR inhibitor rapamycin resulted in significant reduction of FoxO3 null- but not wild type-derived CFU-Sd12 in lethally irradiated hosts. These unexpected results suggested that AKT/mTOR signaling pathway is specifically overactivated as part of a feedback loop mechanism and mediates enhanced generation of FoxO3 null primitive multipotential hematopoietic progenitors in vivo. We further showed that phosphorylation of AKT/mTOR/S6K is highly sensitive to ROS scavenger N-Acetyl-Cysteine (NAC) in vivo and ex vivo in both wild type and FoxO3 null primitive hematopoietic progenitors indicating that ROS are involved in cytokine signaling in primary hematopoietic progenitor cells. Interestingly, in vivo administration of NAC normalized the number of FoxO3 null-derived CFU-Sd12 in lethally irradiated hosts without any impact on wild type CFU-Sd12 strongly suggesting that ROS mediate specifically enhanced generation of primitive hematopoietic progenitors in FoxO3 null mice. In this context, we were surprised to find similar levels of ROS concentrations in FoxO3 mutant as compared to control hematopoietic progenitors. Thus, we asked whether the increase in FoxO3 null primitive hematopoietic progenitor compartment is due to an increase sensitivity of cytokine signaling to ROS as opposed to increased ROS build up per se in these cells. In search for a mechanism we found the expression of Lnk, a negative regulator of cytokine signaling, to be highly reduced in FoxO3 null primitive hematopoietic progenitor cells. We further demonstrated that retroviral reintroduction of Lnk but not vector control in FoxO3 null primitive bone marrow cells reduced significantly the number of FoxO3 null-derived CFU-Sd12in vivo. Collectively, these results suggest that reduced expression of Lnk hypersensitizes FoxO3-deficient hematopoietic progenitors to ROS generated by cytokine signaling leading to myeloproliferation. These cumulative findings uncover a mechanism by which deregulation of cellular sensitivity to physiological ROS leads to hematopoietic malignancies specifically in disorders in which FoxO play a role.

Sign in / Sign up

Export Citation Format

Share Document