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.

Novel Lentiviral Vectors Displaying ‘Early-Acting-Cytokines’ Preferentially Promote the Survival and Transduction of NOD/SCID Repopulating Human Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2107-2107
Author(s):  
E.L.S. Verhoeyen ◽  
Maciej Wiznerowicz ◽  
Delphine Olivier ◽  
Brigitte Izac ◽  
Didier Trono ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vectors ◽  
Hematopoietic Stem ◽  
Efficient Gene ◽  
Cd34 Cells

Abstract A major limitation of current generation lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent target cells which hampers their application for hematopoietic stem cell gene therapy. Human CD34+ cells that reside into G0 phase of the cell cycle and thus are quiescent, are indeed higly enriched in hematopoietic stem cells. Here, we designed novel lentiviral vectors that overcome this type of restriction by displaying early-acting-cytokines on their surface. Presentation of a single cytokine, thrombopoietin (TPO), or co-presentation of TPO and stem cell factor (SCF) on the lentiviral vector surface improved gene transfer into quiescent CD34+ cord blood cells by 45-fold and 77-fold, respectively, as compared to conventional lentiviral vectors. Moreover, these new LVs preferentially transduced and promoted the survival of immature resting cells rather than cycling CD34+ cells. Most importantly, the new early-cytokine-displaying lentiviral vectors allowed highly efficient gene transfer in CD34+ immature cells with long-term in vivo NOD/SCID mice repopulating capacity, a hallmark of bona fide HSCs. In conclusion, the novel ‘early-acting cytokines’ displaying LVs described here provide simplified, reproducible gene transfer protocols that ensure efficient gene transfer in hematopoietic stem cells. As such, these novel reagents bring us one step closer to selective in vivo gene therapy.

scholarly journals Gene transfer into hematopoietic stem cells: long-term maintenance of in vitro activated progenitors without marrow ablation.

1994 ◽  
Vol 91 (1) ◽  
pp. 350-354 ◽  
Author(s):  
D. Bienzle ◽  
A. C. Abrams-Ogg ◽  
S. A. Kruth ◽  
J. Ackland-Snow ◽  
R. F. Carter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Marrow Ablation ◽  
Term Maintenance

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<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 <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.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate

2021 ◽  
Vol 9 ◽  
Author(s):  
Wanbo Tang ◽  
Jian He ◽  
Tao Huang ◽  
Zhijie Bai ◽  
Chaojie Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Hematopoietic Stem ◽  
Genetic Lineage Tracing

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ 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.

Reversible Expression of CD34 by Murine Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2548-2554 ◽  
Author(s):  
Takashi Sato ◽  
Joseph H. Laver ◽  
Makio Ogawa
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Adult Mice ◽  
Cd34 Expression ◽  
Marrow Cells

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34−. We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34− and CD34+ cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage−c-kit+ Sca-1+ CD34− bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34− and CD34+ cell populations and tested for their engraftment capabilities, only CD34+ cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34+ stem cells to CD34− state. We transplanted Ly-5.1 CD34+post–5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34− cell fraction, indicating the reversion of CD34+ to CD34−stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.

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