Subacute AMD3100 Treatment Is Not Efficient in Neonatal Hypoxic-Ischemic Rats

Stroke ◽  
2021 ◽  
Author(s):  
Daiane Aparecida Spiess ◽  
Raquel Maria Pereira Campos ◽  
Luciana Conde ◽  
Nadine Didwischus ◽  
Johannes Boltze ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Brain Atrophy ◽  
Water Maze ◽  
Motor Coordination ◽  
Separate Analysis ◽  
Therapeutic Effects ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Cell Therapies ◽  
Combining Data

Background and Purpose: Despite the advances in treating neonatal hypoxic-ischemic encephalopathy (HIE) with induced hypothermia, the rates of severe disability are still high among survivors. Preclinical studies have indicated that cell therapies with hematopoietic stem/progenitor cells could improve neurological outcomes in HIE. In this study, we investigated whether the administration of AMD3100, a CXCR4 antagonist that mobilizes hematopoietic stem/progenitor cells into the circulation, has therapeutic effects in HIE. Methods: P10 Wistar rats of both sexes were subjected to right common carotid artery occlusion or sham procedure, and then were exposed to hypoxia for 120 minutes. Two subcutaneous injections of AMD3100 or vehicle were given on the third and fourth day after HIE. We first assessed the interindividual variability in brain atrophy after experimental HIE and vehicle treatment in a small cohort of rats. Based on this exploratory analysis, we designed and conducted an experiment to test the efficacy of AMD3100. Brain atrophy on day 21 after HIE was defined as the primary end point. Secondary efficacy end points were cognitive (T-water maze) and motor function (rotarod) on days 17 and 18 after HIE, respectively. Results: AMD3100 did not decrease the brain atrophy in animals of either sex. Cognitive impairments were not observed in the T-water maze, but male hypoxic-ischemic animals exhibited motor coordination deficits on the rotarod, which were not improved by AMD3100. A separate analysis combining data from animals of both sexes also revealed no evidence of the effectiveness of AMD3100 treatment. Conclusions: These results indicate that the subacute treatment with AMD3100 does not improve structural and functional outcomes in a rat HIE model.

scholarly journals Ultrasonic microbubble VEGF gene delivery improves angiogenesis of senescent endothelial progenitor cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yi-Nan Lee ◽  
Yih-Jer Wu ◽  
Hsin-I. Lee ◽  
Hsueh-Hsiao Wang ◽  
Chiung-Yin Chang ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Copy Number ◽  
Therapeutic Effects ◽  
Endothelial Progenitor ◽  
Cell Therapies ◽  
Aortic Endothelial Cells ◽  
Angiogenic Effect ◽  
And Migration

AbstractThe therapeutic effects of ultrasonic microbubble transfection (UMT)-based vascular endothelial growth factor 165 (VEGF165) gene delivery on young and senescent endothelial progenitor cells (EPCs) were investigated. By UMT, plasmid DNA (pDNA) can be delivered into both young EPCs and senescent EPCs. In the UMT groups, higher pDNA-derived protein expression was found in senescent EPCs than in young EPCs. Consistent with this finding, a higher intracellular level of pDNA copy number was detected in senescent EPCs, with a peak at the 2-h time point post UMT. Ultrasonic microbubble delivery with or without VEGF improved the angiogenic properties, including the proliferation and/or migration activities, of senescent EPCs. Supernatants from young and senescent EPCs subjected to UMT-mediated VEGF transfection enhanced the proliferation and migration of human aortic endothelial cells (HAECs), and the supernatant of senescent EPCs enhanced proliferation more strongly than the supernatant from young EPCs. In the UMT groups, the stronger enhancing effect of the supernatant from senescent cells on HAEC proliferation was consistent with the higher intracellular VEGF pDNA copy number and level of protein production per cell in the supernatant from senescent cells in comparison to the supernatant from young EPCs. Given that limitations for cell therapies are the inadequate number of transplanted cells and/or insufficient cell angiogenesis, these findings provide a foundation for enhancing the therapeutic angiogenic effect of cell therapy with senescent EPCs in ischaemic cardiovascular diseases.

Disruption of CXCR4 Utilizing AMD3100 Bypasses Mobilization Deficiency Exhibited by CD26−/− Mice and Results in Efficient Mobilization of Myeloid Progenitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3492-3492
Author(s):  
Laura A. Paganessi ◽  
Andrew L. Walker ◽  
Stephanie A. Gregory ◽  
Henry C. Fung ◽  
Kent W. Christopherson
Keyword(s):  
Bone Marrow ◽  
Blood Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Genetically Engineered Mice ◽  
Colony Forming Units ◽  
Myeloid Progenitors

Abstract The exopeptidase CD26 (also known as DPPIV/dipeptidylpeptidase IV) cleaves dipeptides from the N-terminus of proteins that contain the required X-Pro or X-Ala motif. We have previously reported that inhibition or loss of CD26 activity results in a deficiency in normal granulocyte-colony stimulating factor (G-CSF) induced mobilization, suggesting that CD26 is a necessary component of mobilization (Christopherson, et al Blood 2003 and Christopherson, et al Exp Hematol 2003). The chemokine CXCL12 (SDF-1, stromal cell derived factor-1) contains the appropriate recognition sequence for CD26 induced cleavage. This combined with the importance of CXCL12 in the trafficking of hematopoietic stem and progenitor cells (HSC/HPC) suggests CXCL12 as a likely functional target of CD26 during G-CSF induced mobilization. For this reason we therefore decided to investigate whether genetically engineered mice lacking CD26 (CD26−/−) could be mobilized utilizing the CXCR4 antagonist, AMD3100. To evaluate this, ten week old C57BL/6 and CD26−/− mice (also on a C57BL/6 background) received a single subcutaneous injection of AMD3100 (1mg/1kg). One hour following injection the mice were euthanized by CO2 inhalation. Peripheral blood was then obtained by heart stick with a 1.2 ml syringe containing EDTA as an anticoagulant. A complete blood count was taken for each peripheral blood sample. Following red blood cell lysis, cells were plated for myeloid colony formation in a standard 1% methylcellulose colony assay containing the appropriate cytokines. Following 7 days of incubation at 5% O2, 5% CO2 and 37°C plates were scored for colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte, erythroid, macrophage, and megakaryocytic (CFU-GEMM). Data is presented as the number of colonies per femur for the bone marrow and as the number of colonies per ml of whole blood for the peripheral blood. AMD3100 treatment resulted in an increase in white blood cell (WBC) counts from 5.05±0.48 × 106/ml in untreated mice to 10.21±0.88×106/ml in treated mice (p≤0.01). An increase in WBC counts was also observed during AMD3100 treatment in CD26−/− mice from 7.77±1.28×106/ml in untreated mice to 16.7 ±2.11 × 106/ml in treated mice (p<0.01). AMD3100 treatment resulted in an increase in circulating myeloid progenitors in the peripheral blood of C57BL/6 and CD26−/− mice as compared to untreated C57BL/6 and CD26−/− mice respectively (p≤0.01). Specifically, a 2.38, 3.75, 12.33 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of C57BL/6 mice after treatment. A 2.63, 5.48, 14.29 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of CD26−/− mice after treatment. Existing pre-clinical and clinical data suggest that the CXCR4 antagonist, AMD3100, rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery. The results presented here provide pre-clinical evidence that disruption of the interaction between the CXCR4 chemokine receptor and CXCL12, via sub-cutaneous injection of AMD3100, mobilizes significant numbers of myeloid progenitors in mice, even in the absence of CD26. These results support the notion that CD26 is downstream of G-SCF treatment. Additionally, these results support the potential use of AMD3100 to treat patients that may have an altered ability to respond to G-CSF treatment as a result of a reduction or loss in CD26 activity. Future studies are warranted to evaluate potential variations in CD26 levels or activity in the general population, in differing patient populations, and during different treatment regimens.

Mobilization Studies in Complement-Deficient Mice Reveal That AMD3100 Mobilization Depends On Complement Cascade Activation and Suggest Involvement of “Membrane Attack Complex - MAC” in Egress of Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 367-367
Author(s):  
Marcin Wysoczynski ◽  
HakMo Lee ◽  
Rui Liu ◽  
Wan Wu ◽  
Janina Ratajczak, ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Membrane Attack Complex ◽  
Classical Pathway ◽  
Compensatory Mechanism ◽  
Complement Cascade ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Abstract 367 We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway as seen during G-CSF- or Zymosan mobilization, respectively. As a result, several potent bioactive CC anaphylatoxins (C3 and C5 cleavage fragments) are released that regulate egress of HSPCs (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40, Leukemia 2009; in press.). This explains why: i) NOD/SCID and RAG-/- animals that do not activate the Ig-dependent CC classical pathway; ii) C2fB-/- and C3-/- mice that do not activate the classical and alternative CC pathways; and iii) C5-/- mice that do not activate the distal pathway of CC are all poor G-CSF- and/or Zymosan mobilizers. In this study, we evaluated the role of CC in mobilization induced by CXCR4 antagonist AMD3100. We noticed that all CC activation-deficient mice mentioned above, except C5-/- mice, mobilize normally in response to AMD3100 administration. Accordingly, the number of mobilized CD34- SKL cells, leucocytes, and CFU-GM clonogeneic progenitors in mutant mice was similar to wt littermates. More important we observed that AMD3100 mobilization of HSPCs was preceded by a massive egress of leucocytes from BM and that AMD3100 was able to stimulate in these cells i) phosphorylation of MAPKp42/44 and ii) secretion of MMP-9. At the same time, ELISA data to detect CC activation revealed that serum levels of CC cleavage fragments, which were low in the initial phase of AMD3100 mobilization during granulocyte egress, become elevated later during HSPC egress. Thus, our data show that despite a fact that G-CSF and AMD3100 mobilize HSPCs by involving different mechanisms, activation of CC is a common phenomenon occurring during mobilization induced by both compounds. This further supports a pivotal role of CC activation in the egress of HSPCs from BM; however, both compounds activate CC differently. While G-CSF administration initiates CC activation at its proximal C1q-C3 level, AMD3100 induces CC activation at the distal C5 level, pointing to a crucial role of C5 cleavage in executing mobilization. To support this, all mice employed in our studies that display defects in activation of proximal stages of CC (NOD/SCID, RAG, C2fB-/-, and C3-/-) are normal AMD3100 mobilizers. However, C5 is cleavage required for mobilization occurs in the plasma of these animals latter on - directly by proteases released from AMD3100-stimulated granulocytes that egress from the BM as a first wave of mobilized cells. This compensatory mechanism cannot occur from obvious reasons in C5-/- mice. We conclude that AMD3100-directed mobilization similarly as G-CSF-induced one depends on activation of CC; however, AMD3100 in contrast to G-CSF activates CC at distal stages – directly by proteases released from mobilized/activated granulocytes. Cleavage of C5 and release of C5a and desArgC5a create a sinusoid-permissive environment in BM for HSPCs egress. This suggests involvement of both C5 cleavage fragments as well as a potential role of downstream elements of CC activation - membrane attack complex - MAC (C5b-C9) in stem cell mobilization. Therefore, some poor AMD3100 patient responders could possess a defect in activation of the distal steps of CC. Disclosures: No relevant conflicts of interest to declare.

ALL Cells Mobilized by AMD3100 Are More Proliferative, and Remain In the Circulation Longer Than Normal HSC, Enhancing the Action of Vincristine

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2137-2137 ◽  
Author(s):  
Linda J. Bendall ◽  
Robert Welschinger ◽  
Florian Liedtke ◽  
Carole Ford ◽  
Aileen Dela Pena ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Microenvironment ◽  
Hematopoietic Progenitors ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Cell Mobilization ◽  
Cycle Dependent

Abstract Abstract 2137 The chemokine CXCL12, and its receptor CXCR4, play an essential role in homing and engraftment of normal hematopoietic cells in the bone marrow, with the CXCR4 antagonist AMD3100 inducing the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. We have previously demonstrated that AMD3100 similarly induces the mobilization of acute lymphoblastic leukemia (ALL) cells into the peripheral blood. The bone marrow microenvironment is thought to provide a protective niche for ALL cells, contributing to chemo-resistance. As a result, compounds that disrupt leukemic cell interactions with the bone marrow microenvironment are of interest as chemo-sensitizing agents. However, the mobilization of normal hematopoietic stem and progenitor cells may also increase bone marrow toxicity. To better evaluate how such mobilizing agents affect normal hematopoietic progenitors and ALL cells, the temporal response of ALL cells to the CXCR4 antagonist AMD3100 was compared to that of normal hematopoietic progenitor cells using a NOD/SCID xenograft model of ALL and BALB/c mice respectively. ALL cells from all 7 pre-B ALL xenografts were mobilized into the peripheral blood by AMD3100. Mobilization was apparent 1 hour and maximal 3 hours after drug administration, similar to that observed for normal hematopoietic progenitors. However, ALL cells remained in the circulation for longer than normal hematopoietic progenitors. The number of ALL cells in the circulation remained significantly elevated in 6 of 7 xenografts examined, 6 hours post AMD3100 administration, a time point by which circulating normal hematopoietic progenitor levels had returned to baseline. No correlation between the expression of the chemokine receptor CXCR4 or the adhesion molecules VLA-4, VLA-5 or CD44, and the extent or duration of ALL cell mobilization was detected. In contrast, the overall motility of the ALL cells in chemotaxis assays was predictive of the extent of ALL cell mobilization. This was not due to CXCL12-specific chemotaxis because the association was lost when correction for background motility was undertaken. In addition, AMD3100 increased the proportion of actively cells ALL cells in the peripheral blood. This did not appear to be due to selective mobilization of cycling cells but reflected the more proliferative nature of bone marrow as compared to peripheral blood ALL cells. This is in contrast to the selective mobilization of quiescent normal hematopoietic stem and progenitor cells by AMD3100. Consistent with these findings, the addition of AMD3100 to the cell cycle dependent drug vincristine, increased the efficacy of this agent in NOD/SCID mice engrafted with ALL. Overall, this suggests that ALL cells will be more sensitive to effects of agents that disrupt interactions with the bone marrow microenvironment than normal progenitors, and that combining agents that disrupt ALL retention in the bone marrow may increase the therapeutic effect of cell cycle dependent chemotherapeutic agents. Disclosures: Bendall: Genzyme: Honoraria.

scholarly journals The novel CXCR4 antagonist POL5551 mobilizes hematopoietic stem and progenitor cells with greater efficiency than Plerixafor

Leukemia ◽  
2013 ◽  
Vol 27 (12) ◽  
pp. 2322-2331 ◽  
Author(s):  
D Karpova ◽  
K Dauber ◽  
G Spohn ◽  
D Chudziak ◽  
E Wiercinska ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
The Novel ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

CXCR4 antagonist (BL-8040) to enhance antitumor effects by increasing tumor infiltration of antigen-specific effector T-cells.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 73-73 ◽  
Author(s):  
Pankaj Gaur ◽  
Vivek Verma ◽  
Seema Gupta ◽  
Ella Sorani ◽  
Abi Vainstein Haras ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Tumor Growth ◽  
Progenitor Cells ◽  
Specific Antigen ◽  
Mouse Tumor ◽  
Cxcr4 Antagonist ◽  
Antitumor Effects ◽  
Hematopoietic Stem ◽  
Effector Cells

73 Background: C-X-C chemokine receptor type 4 (CXCR4) helps to retain the hematopoietic stem cells (HSC) in the bone marrow (BM). CXCR4 binds to its ligand CXCL12/SDF1 which is constitutively expressed in the BM thereby inhibiting the mobilization of CXCR4 expressing immune progenitor cells. Moreover, increased numbers of effector cells in the tumor microenvironment (TME) are directly correlated to enhanced immunotherapeutic efficacy. Therefore, we hypothesized that CXCR4 antagonism will result in movement of immune progenitor cells from BM to periphery leading to increased availability of T-cells in the periphery and better infiltration of effector cells into the TME. Methods: In TC-1 mouse tumor-model, we tested the effects of CXCR4 antagonist (BL-8040; 4 doses; 24 h apart; 20 mg/kg) on the tumor growth and mice survival in the presence of tumor-specific antigen priming using E7 peptide vaccine (3 doses, one week apart). Anti-tumor immune responses were determined in the tumor tissues obtained 3-4 days after the second vaccination using flow cytometry. Results: We found that BL-8040 given with specific antigenic stimulation results in significantly enhanced anti-tumor immune response, leading to a decrease in tumor growth (p≤0.001 at day 21) and prolonged mice survival. At day 35 after tumor implantation, 80% of mice survived in the BL-8040 + vaccine treatment group compared to 0% survival following BL-8040 or vaccine treatments. Interestingly, we also found that BL-8040 leads to a significant increase in the numbers of antigen-specific CD8+ T-cells in the TME. We also found that starting BL-8040 prior to or together with the priming of the mice did not affect the outcome, suggesting that the scheduling of BL-8040 does not affect the therapeutic outcomes. Conclusions: These results suggest that BL-8040 treatment enhances anti-tumor immune response by potentially increasing the immune progenitor cells in the periphery leading to a better immune response. These results also suggest that BL-8040, a CXCR4 antagonist, is a promising immune-modulatory agent with potent anti-tumor effects.

scholarly journals Oncostatin M Is a Novel Niche Factor That Restrains Hematopoietic Stem Cell Mobilization in Response to G-CSF and CXCR4 Antagonist Plerixafor

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4469-4469
Author(s):  
Kavita Bisht ◽  
Crystal McGirr ◽  
Kylie A Alexander ◽  
Whitney Fleming ◽  
Natalie Sims ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Stromal Cells ◽  
Receptor Gene ◽  
Fold Increase ◽  
Oncostatin M ◽  
Distribution Analysis ◽  
Mesenchymal Progenitor Cells ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem

Granulocyte colony-stimulating factor (G-CSF) and the CXCR4 chemokine receptor antagonist Plerixafor are used to mobilize hematopoietic stem and progenitor cells (HSPC). However, in the autologous setting, prior chemotherapy and radiotherapy impair HSPC mobilizing response to G-CSF and Plerixafor thereby precluding autologous HSC transplantation. We now demonstrate in mice that oncostatin M (OSM) production in the bone marrow (BM) is dramatically increased in response to G-CSF and that OSM counteracts the mobilizing effect of G-CSF and Plerixafor indirectly through the BM stroma. Indeed, by immunohistochemistry, a 4-day G-CSF treatment strongly increased OSM protein expression in cell clusters containing F4/80+ macrophages in the endosteal and central region of the mobilized BM. OSM concentration was 7-fold higher in BM fluids of G-CSF mobilized mice compared to control mice as measured by ELISA. To investigate OSM effects on HSPC mobilization, C57BL/6 mice (WT) and mice lacking the OSM receptor gene (OSMR-/-) were treated with 125 μg/kg human G-CSF bidaily for 2-6 days and/or Plerixafor (10 mg/kg) one hour before harvest. Absence of OSMR increased HSPC mobilization with a 4-fold increase in colony forming cells (CFC), phenotypic Lin-Kit+Sca1+ HSPC (LKS+ cells) and LKS+Flt3-CD48-CD150+ HSC into the blood at days 2 and 4 of G-CSF treatment and increased accumulation of HSPC and HSC in the spleen of OSMR-/- mice at day 6 of G-CSF treatment. In a competitive repopulation assay with serial dilutions of mobilized blood, deletion of OSMR gene enhanced ≈4-fold mobilization of functional reconstituting HSC in response to G-CSF as shown by Poisson's distribution analysis. HSPC mobilization in response to Plerixafor was also significantly enhanced with a doubling of CFC, HSPC and HSC into the blood of OSMR-/- mice compared to WT. OSMR gene deletion also significantly enhanced HSPC mobilization (3-fold) in response to G-CSF plus Plerixafor combination, the most effective mobilization regimen currently approved. To further demonstrate that endogenous OSM restrains HSPC mobilization in response to G-CSF, we produced a recombinant mouse OSM-trap, a dimeric protein chimera made of the extracellular domain of mouse OSMR and mouse gp130 fused with a mutant Fc fragment of mouse IgG2a. Co-administration of this OSM-trap together with G-CSF also increased HSPC and HSC mobilization into the blood in response to G-CSF compared to mice treated with G-CSF and control trap construct. This negative effect of endogenous OSM on HSPC mobilization was indirectly mediated by BM stromal cells. qRT-PCR on BM myeloid and stromal cells sorted from steady-state or mobilized mice showed that Osm mRNA is mostly expressed by CD11b+F4/80+CD169+VCAM1+ macrophages and CD11b+F4/80-Ly6G+ neutrophils. In sharp contrast, Osmr mRNA was undetectable in any sorted BM leukocyte population, but abundantly expressed by CD45-Lin-CD31+Sca1+CD105+ endothelial and CD45-Lin-CD31-CD51+PDGFRα+/-Sca-1+/- mesenchymal progenitor cells. OSMR protein was expressed by vascular beds in the BM as detected by IHC. Confirming the indirect effect of OSM, we found that recombinant OSM had no effect on HSPC chemotaxis in response to CXCL12, or HSPC adhesion to immobilized VCAM-1 or selectins in vitro. However, HSPC freshly isolated from the BM of OSMR-/- mice had enhanced chemotaxis in response to CXCL12 compared to HSPC isolated from WT mice. In conclusion, G-CSF increases OSM expression in the BM, and this appears to restrain HSPC mobilization in response to G-CSF via OSMR-expressing endothelial and mesenchymal cells that decreases HSPC responsiveness to CXCL12 gradient. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Levesque:GlycoMimetics: Equity Ownership.

Precise and Efficient Crispr/Cas9 Mediated Gene Editing in Long-Term Engrafting Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2312-2312
Author(s):  
Jack M Heath ◽  
Aditi Chalishazar ◽  
Christina S Lee ◽  
William Selleck ◽  
Cecilia Cotta-Ramusino ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Gene Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Hematopoietic Stem ◽  
Cell Therapies ◽  
Immunodeficient Mice ◽  
Cd34 Cells

Abstract Transplantation of gene-modified autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for several hematologic diseases. However, a number of blood disorders may not be amenable to gene augmentation-based therapeutics. Targeted genome editing in human HSPCs could provide a therapeutic approach for these otherwise untreatable diseases. Here we demonstrate that CRISPR/Cas9 ribonucleoprotein (RNP) edits target genes in human HSPCs with high efficiency and precision. Human adult and umbilical cord blood (CB) CD34+ cells from 20 donors were electroporated with S. pyogenes or S. aureus Cas9 RNP targeting HBB, AAVS1, or CXCR4. Sequence analysis demonstrated up to 80% editing in CB CD34+ cells (mean±s.d: 61%±9%) and up to 57% in adult CD34+ cells (39%±13%). Delivery of Cas9 RNP and a single-stranded oligodeoxynucleotide donor (ssODN) led to up to 12% ssODN-mediated homology directed repair (HDR) and also led to a 20% increase in total gene editing (HDR+NHEJ)(RNP: 48%±15%; RNP+ssODN: 69%±8%). Both Cas9 RNP gene-edited CD34+ cells and donor-matched untreated control CD34+ cells reconstituted human hematopoiesis in primary and secondary recipient immunodeficient mice, with ~85% human CD45+ cell peripheral blood reconstitution 4 months after primary transplantation. Human T and B lymphoid, erythroid, and myeloid cells were detected in the spleen, thymus, and bone marrow with 20% CD34+ cell engraftment in the marrow of mice transplanted with RNP gene-edited or control CD34+ cells. The level of targeted gene editing in human erythroid, myeloid, and CD34+ cells that were recovered and enriched from the hematopoietic organs of primary recipients (~50%) was similar to the level of gene editing detected in the pre-infusion product (~60%). In summary, these results indicate that Cas9 gene-edited human HSPCs retain long-term engraftment potential and support multilineage blood reconstitution in vivo, thus supporting further investigation of CRISPR/Cas9 mediated gene-edited hematopoietic stem/progenitor cell therapies. Disclosures Heath: Editas Medicine: Employment. Chalishazar:Editas Medicine: Employment. Lee:Editas Medicine: Employment. Selleck:Editas Medicine: Employment. Cotta-Ramusino:Editas Medicine: Employment. Bumcrot:Editas Medicine: Employment. Gori:Editas Medicine: Employment.

scholarly journals Regulatory Systems in Bone Marrow for Hematopoietic Stem/Progenitor Cells Mobilization and Homing

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
P. Alvarez ◽  
E. Carrillo ◽  
C. Vélez ◽  
F. Hita-Contreras ◽  
A. Martínez-Amat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Proteolytic Enzymes ◽  
Influential Factors ◽  
Hematopoietic Stem ◽  
Cell Therapies ◽  
Cord Injury ◽  
Cell Release ◽  
Stromal Cell Derived Factor

Regulation of hematopoietic stem cell release, migration, and homing from the bone marrow (BM) and of the mobilization pathway involves a complex interaction among adhesion molecules, cytokines, proteolytic enzymes, stromal cells, and hematopoietic cells. The identification of new mechanisms that regulate the trafficking of hematopoietic stem/progenitor cells (HSPCs) cells has important implications, not only for hematopoietic transplantation but also for cell therapies in regenerative medicine for patients with acute myocardial infarction, spinal cord injury, and stroke, among others. This paper reviews the regulation mechanisms underlying the homing and mobilization of BM hematopoietic stem/progenitor cells, investigating the following issues: (a) the role of different factors, such as stromal cell derived factor-1 (SDF-1), granulocyte colony-stimulating factor (G-CSF), and vascular cell adhesion molecule-1 (VCAM-1), among other ligands; (b) the stem cell count in peripheral blood and BM and influential factors; (c) the therapeutic utilization of this phenomenon in lesions in different tissues, examining the agents involved in HSPCs mobilization, such as the different forms of G-CSF, plerixafor, and natalizumab; and (d) the effects of this mobilization on BM-derived stem/progenitor cells in clinical trials of patients with different diseases.

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