scholarly journals 690. Culture of Mobilized Rhesus Macaque CD34+ Cells in Hypoxic Conditions Does Not Improve Lentiviral Transduction Efficiency in Long-Term Repopulating Hematopoietic Stem Cells

2014 ◽  
Vol 22 ◽  
pp. S267
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Rhesus Macaque ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Lentiviral Transduction ◽  
Hypoxic Conditions ◽  
Cd34 Cells

scholarly journals AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3772-3778 ◽  
Author(s):  
André Larochelle ◽  
Allen Krouse ◽  
Mark Metzger ◽  
Donald Orlic ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Genetic Manipulation ◽  
Retroviral Vectors ◽  
Lymphoid Cells ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Cd34 Cells

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34+ cells were phenotypically analyzed, marked with NeoR-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34+ cells with NeoR gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34+ cells are in the G1 phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34+ cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34+ cells were better than those obtained using CD34+ cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34+ cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

scholarly journals Effect of Mesenchymal Stem Cell-derived Microvesicles on Megakaryocytic Differentiation of CD34+ Hematopoietic Stem Cells

2020 ◽  
Vol 10 (2) ◽  
pp. 315-322
Author(s):  
Sara Aqmasheh ◽  
Karim Shamsasenjan ◽  
Elham Khalaf Adeli ◽  
Aliakbar Movassaghpourakbari ◽  
Parvin Akbarzadehlaleh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Significant Expression ◽  
Late Stages ◽  
Megakaryocyte Differentiation

Purpose: Mesenchymal stem cells (MSCs) release hematopoietic cytokines, growth factors, and Microvesicles (MVs) supporting the hematopoietic stem cells (HSCs). MVs released from various cells, playing a crucial role in biological functions of their parental cells. MSC-derived MVs contain microRNAs and proteins with key roles in the regulation of hematopoiesis. Umbilical cord blood (UCB) is a source for transplantation but the long-term recovery of platelets is a main problem. Therefore, we intend to show that MSC-MVs are able to improve the differentiation of UCB-derived CD34+ cells to megakaryocyte lineage. Methods: In this descriptive study, MSCs were cultured in DMEM to collect the culture supernatant, which was ultracentrifuged for the isolation of MVs. HSCs were isolated from UCB using MACS method and cultured in IMDM supplemented with cytokines and MVs in three different conditions. Megakaryocyte differentiation was evaluated through the expression of specific markers and genes after 72 hours, and the data was analyzed by t test (P<0.05). Results: The expression of specific megakaryocyte markers (CD41 and CD61) in the presence of different concentrations of MSC-MVs did not show any significant difference. Also, the expression of specific genes of megakaryocyte lineage was compared with control group. The expression of GATA2 and c-Mpl was significantly increased, GATA1 was not significantly decreased, and FLI1 was significantly decreased. Conclusion: MSC-MVs could improve the expression of specific megakaryocyte genes; however, there was no significant expression of CD markers. Further studies, including the evaluation of late stages of megakaryocyte differentiation, are required to evaluate platelet production and shedding

Suicide Gene (HSVtk) Ablation of Hematopoietic Stem Cells and Their Progeny In the Rhesus Macaque Model: An Approach to Deletion of Dangerous Clones.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3759-3759
Author(s):  
Cecilia Barese ◽  
Connor King ◽  
Stephanie Sellers ◽  
Allen E Krouse ◽  
Mark E Metzger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Hematopoietic Stem Cells ◽  
Rhesus Macaque ◽  
Conflicts Of Interest ◽  
Marker Gene ◽  
Suicide Gene ◽  
Marker Genes ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3759 For genetic blood diseases, such as primary immunodeficiencies, gene therapy targeted to hematopoietic stem cells (HSCs) is a feasible and now proven effective therapeutic option for patients who lack a histocompatible HSC. However, the risk of adverse outcomes resulting from insertional oncogenesis is a major concern. We are investigating whether inclusion of the herpes simplex virus thymidine kinase (HSVtk) gene into integrating vectors into rhesus macaque HSCs confers ganciclovir (GCV) sensitivity allowing ablation of vector-containing cells from the blood and other hematopoietic compartments, as an approach to increasing safety of gene therapy procedures. HSVtk suicide genes have been studied in detail in transduced mature T cells, but never in stem and progenitor cells. We infused autologous CD34+ cells transduced ex vivo with gammaretrovirus vectors encoding the HSVtk as suicide gene along with marker genes into 4 rhesus macaques, following myeloablative irradiation. In the first animal, a vector consisting of the MND backbone driving the sr39 high affinity tk mutant, and IRES and a truncated NGFR marker gene was used. A stable marking level of 5% NGFR+ circulating cells was observed for 6 months following transplantation, confirmed by q-PCR. The drug GCV was infused at 5 mg/Kg BID for 21 days. This animal had complete elimination of vector-containing cells in all peripheral blood lineages as assessed by flow cytometry and qPCR, and remains negative now 4 months after GCV discontinuation. Three additional animals were transplanted with autologous CD34+ cells transduced with a vector containing a standard HSVtk gene and GFP as a marker. These animals had lower stable marking levels of approximately 1% at 4 months post-transplant, and after 21 days of GCV, had a clear decrease in the level of GFP+ cells, but not complete ablation, likely due to lower drug-sensitivity of the tk protein expressed by this vector. Cells with a lower level of GFP expression were not eliminated, supporting this hypothesis. Additional animals receiving cells transduced with the sr39 tk retroviral vector and with a lentiviral vector containing a codon-optimized HSVtk are in progress. These data suggest that inclusion of a suicide gene in integrating vectors may be an effective way to address genotoxicity concerns, should clonal outgrowth occur, and increase safety of HSC-targeted gene therapy. Disclosures: No relevant conflicts of interest to declare.

Robo4/Magic Roundabout Is a Novel Surface Marker for Murine and Human Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 682-682
Author(s):  
Fumi Shibata ◽  
Yuko Goto-Koshino ◽  
Miyuki Ito ◽  
Yumi Fukuchi ◽  
Yoshihiro Morikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Surface Markers ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cd34 Cells ◽  
Human Hscs

Abstract A variety of cell surface markers such as c-Kit, Sca-1, CD34 and Flt-3 have been utilized to prospectively isolate murine or human hematopoietic stem cells (HSCs). While murine HSCs were shown to be highly enriched in CD34−c-Kit+Sca-1+Lineage- (CD34−KSL) fraction, this population is still not homogeneous for long-term HSCs. In human, CD34+ cells are regarded as crude HSC fraction and used for clinical applications. However, quiescent human HSCs are also found in CD34− fraction, indicating that CD34 is not a bona fide marker for human HSC. Thus, novel surface markers that can be used to purify human or murine HSCs to homogeneity need to be identified. Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are predominantly expressed in nervous system. Slit2, a ligand for Robo, is a large leucine-rich repeat-containing secreted protein that is also expressed in brain. By binding with Robo, Slit2 acts as a repellant for axon guidance of developing neurons and they are critical for correct wiring of neuronal network. Robo family comprises four family members, Robo1 – Robo4, and Robo4 is distinct in that it is expressed specifically in endothelial cells, but not in brain. In this study, we investigated Robo4 for its possible application for HSC identification in murine and human hematopoietic system. By RT-PCR, Robo4 was specifically expressed in murine KSL fraction, and was not expressed in lineage positive cells and various progenitors such as common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), megakaryocyte/erythroid progenitor (MEP) and common lymphoid progenitor (CLP). Moreover, the expression of Robo4 was highest in side population of KSL cells (KSL-SP), and moderate in KSL-main population (KSL-MP) cells. Monoclonal antibody raised against Robo4 identified its high expression in KSL cells by FACS. FACS analysis of human cord blood cells revealed that Robo4 is highly expressed in CD34+ cells, and CD34+Robo4high population fell into CD38− fraction, which enriches human HSCs. Bone marrow transplantation experiments revealed that Robo4+ fraction of murine KSL cells had long-term repopulating activity, while Robo4−KSL cells not. Although both Robo4+ and Robo4− CD34−KSL cells repopulated murine hematopoietic system for long-term, Robo4+CD34−KSL cells achieved higher chimerism after repopulation compared with Robo4−CD34−KSL. To investigate the physiological role of Robo4 in HSC homeostasis, we next examined the expression of Slit2 in hematopoietic system. Interestingly, Slit2 is specifically expressed in bone marrow stromal cells, but not in hematopoietic cells. Moreover, Slit2 is induced in osteoblasts, a critical cellular component composing HSC niche, in response to myelosuppressive stress such as 5FU treatment. These results indicate that Robo4 is expressed in murine and human hematopoietic HSCs and useful for HSC purification, and Robo4 - Slit2 system may play a role in HSC physiology in niche environment under hematopoietic stress.

Novel lentiviral vectors displaying “early-acting cytokines” selectively promote survival and transduction of NOD/SCID repopulating human hematopoietic stem cells

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3386-3395 ◽  
Author(s):  
Els Verhoeyen ◽  
Maciej Wiznerowicz ◽  
Delphine Olivier ◽  
Brigitte Izac ◽  
Didier Trono ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Surface Display ◽  
Lentiviral Vectors ◽  
Hematopoietic Stem ◽  
Efficient Gene ◽  
Cd34 Cells

AbstractA major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells, such as human CD34+ cells, that reside in the G0 phase of the cell cycle and that are highly enriched in hematopoietic stem cells. This hampers their application for gene therapy of hematopoietic cells. Here, we designed novel LVs that overcome this restriction by displaying “early-acting cytokines” on their surface. Display of thrombopoietin, stem cell factor, or both cytokines on the LV surface allowed efficient gene delivery into quiescent cord blood CD34+ cells. Moreover, these surface-engineered LVs preferentially transduced and promoted survival of resting CD34+ cells rather than cycling cells. Finally, and most importantly, these novel LVs allowed superior gene transfer in the most immature CD34+ cells as compared to conventional LVs, even when the latter vectors were used to transduce cells in the presence of recombinant cytokines. This was demonstrated by their capacity to promote selective transduction of CD34+ cell in in vitro derived long-term culture-initiating cell (LTC-IC) colonies and of long-term NOD/SCID repopulating cells (SRCs) in vivo.

scholarly journals Etoposide-Initiated MLL Rearrangements Detected at High Frequency in Human Primitive Hematopoietic Stem Cells with In Vitro and In Vivo Long-Term Repopulating Potential.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 98-98 ◽  
Author(s):  
Jolanta Libura ◽  
Marueen Ward ◽  
Grzegorz Przybylski ◽  
Christine Richardson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Scid Mouse ◽  
P Value ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Rearrangements involving the MLL gene locus at chromosome band 11q23 are observed in therapy-related acute myeloid leukemia and myelodysplastic syndromes following treatment with topoisomerase II (topoII) inhibitors including etoposide. We have shown that one hour of etoposide exposure (20–50 μM) stimulates stable MLL rearrangements in primary human CD34+ cells and that the spectrum of repair products within MLL gene is broader than so far described (Libura et al, Blood, 2005). Clinical data suggest that MLL-associated malignant leukemias originate within primitive hematopietic stem cells capable of differentiation into all hematopoietic lineages and repopulation of myelo-ablated hosts. These cells can be analyzed using the in vivo NOD-SCID mouse model as well as the in vitro long-term culture initiating cell (LTC-IC) assay. We adopted our in vitro CD34+ cell culture model to investigate the impact of etoposide exposure on the most primitive hematopoietic stem cells using parallel assays for LTC-IC and NOD-SCID Repopulating Cells (SRC). Following etoposide exposure (20–50 μM for 1 hour), and 48–96 hours recovery in vitro, untreated control and etoposide-treated CD34+ cells were either seeded in LTC-IC with a supportive feeder layer (Stem Cell Technologies, Inc.), or injected into NOD-SCID mice (0.1–1.5x106 cells per mouse). After 12 weeks, both LTC-IC cultures and bone marrow cells from NOD-SCID mice were seeded in methylcellulose media supplemented with growth factors that promote only human cell colony formation. An increased number of colonies in etoposide-treated samples was obtained from LTC-IC cultures in 3 out of 5 experiments (p value&lt;0.05). This increase in colony number was more dramatic in etoposide-treated samples from NOD-SCID bone marrow (57 versus 0, 8 versus 0). These data demonstrate that etoposide exposure can significantly alter the potential of early hematopoietic stem cells to survive and proliferate both in vitro and in vivo. Injection of as few as 3x105 CD34+ cells into a NOD-SCID mouse was sufficient to obtain methylcellulose colonies, suggesting that this method can be used for the analysis of cells obtained from a single patient sample. Mutation analysis of human methylcellulose colonies derived from both LTC-IC and NOD-SCID was performed by inverse PCR and ligation-mediated PCR followed by sequencing. This analysis revealed that rearrangements originating within the MLL breakpoint cluster region (bcr) were present in 12 out of 29 colonies from etoposide-treated samples versus 5 out of 39 colonies from control samples (p value &lt;0.01), demonstrating that etoposide exposure promotes stable rearrangements within a hematopoietic stem cell compartment with significant proliferative potential. Eight of the 17 events were sequenced, and showed 6 MLL tandem duplications within intron 8, one complex translocation between MLL and chr.15 and tandem duplication, and one event with foreign sequence of unknown origin. Our data are the first report of the spectrum and frequency of MLL rearrangements following topo II inhibitor exposure in a cell population thought to be the target for recombinogenic events leading to therapy-related leukemias.

Effects of Cyclosporine A on Lentiviral Transduction of Mouse Hematopoietic Stem Cells and Transplantation Using Nonmyeloablative Conditioning.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5147-5147
Author(s):  
Nadia D. Sutherland ◽  
H. Trent Spencer
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cyclosporine A ◽  
Fold Increase ◽  
Lentiviral Vectors ◽  
Cell Isolation ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Lentiviral Transduction ◽  
Cells Cultured

Achieving high level transduction of murine hematopoietic stem cells (HSCs) using lentiviral vectors has been a challenge for many laboratories. We investigated the efficiency of lentiviral transduction of murine stem cell antigen-1+ (sca-1+) cells with and without cyclosporine A (CSA), which has previously been shown to increase the transduction efficiency of other types of murine cells. Sca-1+ cells were isolated from C57BL/6 mice and transduced with lentiviral vectors encoding green fluorescent protein (GFP) at various multiplicity of infections (MOI) and with various concentrations of CSA. Twenty-four hours after a single transduction, 1.5 x 104 or 4.5 x 104 cells were plated in methylcellulose containing the appropriate cytokines for progenitor cell growth, and colonies were counted on days 8–12. In the absence of CSA only 4± 2% of progenitor colonies were GFP+. However, when CSA (10 μM) was added within 3 hours of sca-1 cell isolation, transduction efficiency increased to 44 ± 6%. Although the transduction efficiency increased 10-fold, the number of progenitor colonies significantly decreased when CSA was added (up to 90% decrease). Lower concentrations of CSA (e.g. 1 μM) were less toxic to sca-1+ cells but resulted in inconsistent transduction efficiencies. We next determined the effects of CSA when applied at various times after sca-1 cell isolation. We found that the number of sca-1+ cells decreased within the first two days of culture but then begin to increase on day 3, and by day 7 there is a 7-fold increase compared to the number of cells originally isolated. Cells cultured with virus alone had an average increase of 3.5-fold on day 7, but only 3% of cells cultured in CSA survived to day 7. Cells cultured with both virus and CSA had no viability on day 7. However, by delaying the addition of virus and CSA until day 3, a 1.4-fold increase in sca-1+ cells was observed by day 7, which was achieved without affecting the efficiency of transduction. Sca-1+ cells were then transduced with the lentiviral vector in the presence of CSA and transplanted into transgenic sickle mice using a nonmyeloablative conditioning regimen that consisted of busulfan (25 mg/kg) administered on day -1 and costimulation blockade with CTLA-4Ig and anti-CD40 ligand administered on days 0, 2, 4, and 7. We were able to achieve donor engraftment levels of 98% with a 40% engraftment of gene-modified cells. These results show that using CSA in lentiviral transductions of murine HSCs can be an effective method for increasing overall transduction efficiency, and may aid in the use of lentiviral vectors in animal studies.

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