Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3737-3747 ◽  
Author(s):  
Dirk Heckl ◽  
Daniel C. Wicke ◽  
Martijn H. Brugman ◽  
Johann Meyer ◽  
Axel Schambach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Aplastic Anemia ◽  
Bone Marrow Cells ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Egfp Reporter

AbstractThpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl−/−) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl−/− bone marrow cells into Mpl−/− mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl−/− cells had increased long-term repopulating potential, with a marked increase in lineage−Sca1+cKit+ cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage−Sca1+cKit+ cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Activation of AKT1 in Hematopoietic Stem Cells Causes a Myeloproliferative Disease in a Tet-Inducible Mouse Model.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1356-1356
Author(s):  
Christian Brandts ◽  
Miriam Rode ◽  
Beate Lindtner ◽  
Gabriele Koehler ◽  
Steffen Koschmieder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
De Novo ◽  
Myeloproliferative Disorder ◽  
Myeloproliferative Disease ◽  
Hematopoietic Stem ◽  
Akt Phosphorylation

Abstract Activating mutations in Flt3, N- and K-Ras have been reported in all AML subtypes and represent common molecular defects in de novo AML. We have previously shown that these mutations lead to constitutive AKT phosphorylation and activation. As a consequence, Akt phosphorylation is found in myeloid blasts of the majority of AML patients. We reasoned that constitutively active AKT may contribute to leukemia development, and therefore we assessed the contribution of AKT in oncogenic transformation in vivo. For this purpose, we established an inducible mouse model expressing myristylated AKT1 under the control of the scl-3′ enhancer (MyrAKT1). This system restricts activated AKT1 to endothelium, hematopoietic stem cells and myeloid lineage cells at a low but detectable level. About 40% of induced mice developed a myeloproliferative disorder after latencies of 7 to 22 months. Onset of disease was frequently associated with hemangioma formation, due to endothelial MyrAKT1 expression. The myeloproliferative disorder was associated with splenomegaly with increased extramedullary hematopoiesis, while the peripheral blood contained mature granulocytes. Furthermore, the stem cell and progenitor cell compartment in spleens and bone marrow of these mice was altered compared to control mice. Colony formation assays with MyrAKT1-expressing bone marrow suggested that overactivation of AKT1 enhanced proliferation. The AKT1-induced disease was transplantable by both bone marrow and spleen cells. These findings highlight the oncogenic capacity of constitutively activated AKT1 in vivo and indicate that AKT is an attractive target for therapeutic intervention in AML.

scholarly journals Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 337-343 ◽  
Author(s):  
CA Corey ◽  
AD DeSilva ◽  
CA Holland ◽  
DA Williams
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Genetic Sequences ◽  
Marrow Cells

Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate- induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.

scholarly journals Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.

1996 ◽  
Vol 183 (4) ◽  
pp. 1797-1806 ◽  
Author(s):  
M A Goodell ◽  
K Brose ◽  
G Paradis ◽  
A S Conner ◽  
R C Mulligan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Side Population ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Quiescent Stem Cells ◽  
Marrow Cells

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the animal. While experimenting with staining of murine bone marrow cells with the vital dye, Hoechst 33342, we discovered that display of Hoechst fluorescence simultaneously at two emission wavelengths revealed a small and distinct subset of whole bone marrow cells that had phenotypic markers of multipotential HSC. These cells were shown in competitive repopulation experiments to contain the vast majority of HSC activity from murine bone marrow and to be enriched at least 1,000-fold for in vivo reconstitution activity. Further, these Hoechst-stained side population (SP) cells were shown to protect recipients from lethal irradiation at low cell doses, and to contribute to both lymphoid and myeloid lineages. The formation of the Hoechst SP profile was blocked when staining was performed in the presence of verapamil, indicating that the distinctly low staining pattern of the SP cells is due to a multidrug resistance protein (mdr) or mdr-like mediated efflux of the dye from HSC. The ability to block the Hoechst efflux activity also allowed us to use Hoechst to determine the DNA content of the SP cells. Between 1 and 3% of the HSC were shown to be in S-G2M. This also enabled the purification of the G0-G1 and S-G2M HSC had a reconstitution capacity equivalent to quiescent stem cells. These findings have implications for models of hematopoietic cell development and for the development of genetic therapies for diseases involving hematopoietic cells.

scholarly journals Development of a Novel Selective Amplifier Gene for Controllable Expansion of Transduced Hematopoietic Cells

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3884-3892 ◽  
Author(s):  
Keiko Ito ◽  
Yasuji Ueda ◽  
Masaki Kokubun ◽  
Masashi Urabe ◽  
Toshiya Inaba ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Selective Amplifier ◽  
Marrow Cells

Abstract To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)–dependent murine cell line, Ba/F3, the cells showed IL-3–independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Δ(5-195)GCRER, whose product lacks the extracellular G-CSF–binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Δ(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

Identification of Non-Hematopoietic Stem Cells in Mouse Bone Marrow Osteoblastic Zone.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 677-677
Author(s):  
Hidetaka Sato ◽  
Fumio Arai ◽  
Mami Shibata ◽  
Atsushi Hirao ◽  
Toshio Suda ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Fibroblastic Cells ◽  
Marrow Cells

Abstract Bone marrow contains hematopoietic and non-hematopoietic stem cells. Although the character of hematopoietic stem cells (HSCs) in vivo has been extensively studied, our understanding of non-hematopoietic stem cells is derived only from in vitro expanded culture such as mesenchymal stem cells (MSCs). Since the number of non-hematopoietic stem cells in bone marrow is small, their characteristics, anatomical location and physiological role in vivo remain unknown. We attempted to overcome this problem by introducing a novel method. We extracted bone marrow cells from osteoblastic zone by enzymatic digestion. Bone marrow extracted cells (BMECs) contained non-hematopoietic cells (CD45−/Ter-119− cells) more than flushed out bone marrow cells (BMCs). Non-hematopoietic cells in BMECs were divided into two types, CD34+ (BME 34+) cells and CD34− (BME 34−) cells. When BME 34− cells were cultured to expand to a confluent layer in MSC culture condition, they formed fibroblastic cells, which have the ability to differentiate into osteocytes, chondrocytes and adipocytes, indicating that BME 34− cells are the in vivo origin of MSCs. On the other hand, BME CD34+ cells contained mostly non-hematopoietic SP fraction and they were Sca-1+ cells. Immunohistochemical analyses show that CD31-BME 34+ cells localized on osteoblasts and endothelial cells in bone marrow. When BME 34+ cells were co-cultured with stromal (OP9) cells, they formed a colony of myogenic cells, endothelial cells or adipogenic cells. However, different from BME 34− cells, fibroblastic cells expanded from BME 34+ cells did not differentiate to osteocytes, chondrocytes or adipocytes in MSC culture condition. When 4,000-6,000 BME 34+ cells were transplanted into irradiated mice, these cells engrafted as non-hematopoietic Vimentin+ fibroblastic-like cells in various organs such as liver, spleen, pancreas, colon, intestine, kidney, skin, brain, lung, heart, skeletal muscle, and bone marrow. In spleen and liver, transplanted cells formed clusters of Vimentin+ fibroblastic cells indicating that BME 34+ cells have ability to expand in vivo to some extent. When BME 34+ cells were directly injected into tibial muscles which were damaged by cardiotoxin, these cells gave rise to mature skeletal muscle cells and endothelial cells. In summary, these studies show distinguishable two types of non-hematopoietic stem cells in bone marrow. It is possible that mobilization and recruitment of these bone marrow-derived non-hematopoietic stem cells play a major role in the setting of wound healing, organ regeneration and tumor growth. In addition, non-hematopoietic two types of BMECs may contribute to clarify the debate on the stem cell plasticity of bone marrow cells.

scholarly journals Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 337-343 ◽  
Author(s):  
CA Corey ◽  
AD DeSilva ◽  
CA Holland ◽  
DA Williams
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Genetic Sequences ◽  
Marrow Cells

Abstract Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate- induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.

Altered colony-forming activities of bone marrow hematopoietic stem cells in mice following short-term in vivo exposure to parathion

1987 ◽  
Vol 5 (3) ◽  
pp. 231-241 ◽  
Author(s):  
Vincent S. Gallicchio ◽  
Thomas D. Watts ◽  
George P. Casale ◽  
Philip M. Bartholomew
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Short Term ◽  
Hematopoietic Stem

scholarly journals Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 74-83 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Marker Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

scholarly journals Transplantation of hematopoietic stem cells obtained by a combined dye method fractionation of murine bone marrow

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1240-1246 ◽  
Author(s):  
I McAlister ◽  
NS Wolf ◽  
ME Pietrzyk ◽  
PS Rabinovitch ◽  
G Priestley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Light Scatter ◽  
Isolated Cells ◽  
Term Survival ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Separation Media

Abstract Hematopoietic stem cells were purified from murine bone marrow cells (BMC). Their characteristic density, size, internal complexity, Hoechst 33342 dye uptake, and wheat germ agglutinin (WGA) affinity were used to distinguish them from other cells in the bone marrow. BMC suspensions were centrifuged over Ficoll Lymphocyte Separation Media (Organon Teknika, Durham, NC; density 1.077 to 1.08). The lower-density cells were drawn off, stained with Hoechst and labeled with biotinylated WGA bound to streptavidin conjugated to phycoerythrin (WGA-B*A-PE) or with WGA conjugated to Texas Red. These cells were then analyzed and sorted by an Ortho Cytofluorograph 50-H cell sorter. The cells exhibiting medium to high forward light scatter, low to medium right angle light scatter, low Hoechst intensity, and high WGA affinity were selected. Sorted BMC (SBMC) were stained with Romanowsky-type stains for morphologic assay, and were assayed in lethally irradiated (LI) mice for their ability to produce colony-forming units in the spleen (CFU-S) and for their ability to produce survival. The spleen seeding factor for day 8 CFU-S upon retransplantation of the isolated cells was 0.1. The isolated cells were found to have consistent morphology, were enriched up to 135-fold as indicated by day 8 CFU-S assay, 195-fold as indicated by day 14 CFU-S assay, and 150 sorter-selected BMC were able to produce long-term survival in LI mice with retention of donor karyotype. When recipients of this first transplantation were themselves used as BMC donors, their number of day 8 and day 12 CFU-S were found to be reduced. However, 3 X 10(5) of their BMC provided 100% survival among secondary recipients. When the previously SBMC were competed after one transplantation against fresh nonsorted BMC in a mixed donor transplant, they showed the decline in hematopoietic potency normally seen in previously transplanted BMC. We conclude that the use of combinations of vital dyes for fluorescence-activated cell sorting (FACS) selection of survival-promoting murine hematopoietic stem cells provides results comparable with those produced by antibody- selected FACS and has the advantage of a method directly transferable to human BMC.

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