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 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

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.

Stable Polyclonal Murine Long-Term Hematopoiesis without Clonal Exhaustion of MGMT P140K Expressing Murine Hematopoietic Stem Cells after Extended Reduced Intensity Selection.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3271-3271
Author(s):  
Claudia R. Ball ◽  
Manfred Schmidt ◽  
Ingo H. Pilz ◽  
Fessler Sylvia ◽  
David A. Williams ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Hematopoietic Stem ◽  
Reduced Intensity ◽  
Selection Of

Abstract Gene therapy is a promising approach for the therapy of hereditary diseases, but after the occurrence of adverse side effects in a SCID-X1 gene therapy trial increased biological safety has become a major goal of gene therapy. A reduction of the number of transplanted cells could help achieve this goal by reducing the statistical likelihood of insertional mutagenesis simply by simply reducing the number of transplanted cells carrying potentially untoward insertion sites. As we have previously shown, incorporation of the selectable marker gene MGMT P140K into a retroviral vector allows a reduced intensity and toxicity in vivo selection of low numbers of genetically modified hematopoietic cells by chemotherapy with O6-benzylguanine (O6BG) and nitrosourea drugs such as 1,3-bis-2 chloroethyl-1-nitrosourea (BCNU). However, it is still not known whether extended selection over longer periods of time influences the long-term proliferation and differentiation capacity of murine haematopoietic stem cells. To address this question, serial transplantations of murine MGMT-P140K-expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were performed. After ex vivo gene transfer of a MGMT/IRES/eGFP-encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and serially transplanted. First, 2nd and 3rd generation recipient mice were subsequently treated every four weeks in order to amplify treatment effects on the long-term clonal behaviour of modified hematopoietic stem cells. Lineage contribution of transduced hematopoiesis was monitored by FACS over a total of 17 rounds of selection and clonality was monitored by LAM-PCR over a total of 16 rounds of selection. In primary mice, the percentage of transduced blood cells increased from 4.7 ± 0.8 % to 36.4 ± 9.8 % (n=12) and in secondary mice from 29.9 ± 7.2 % to 65.1 ± 8.7 % (n=18) after selection without inducing persistent peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential in the transduced compared to control cells in 1st and 2nd generation animals. LAM PCR analysis of peripheral blood revealed stable oligo- to polyclonal hematopoiesis in 1st, 2nd and 3rd generation mice. Evidence of predominant clones or clonal exhaustion was not observed despite of up to 16 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice which had received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis. This is molecular proof that extensive self-renewal of transplantable stem cells had occurred in the primary mice resulting in a net symmetric refilling of the stem cell compartment. In summary, we demonstrate that even extended selection of MGMT-P140K-expressing hematopoietic stem cells by repetitive chemotherapy does not affect differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies intending to employ amplification of a limited number of genetically modified clones in clinical gene therapy.

scholarly journals Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow.

1992 ◽  
Vol 175 (1) ◽  
pp. 175-184 ◽  
Author(s):  
N Uchida ◽  
I L Weissman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Stem Cell Activity ◽  
Marrow Cells

Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.

scholarly journals BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells

Blood ◽  
2010 ◽  
Vol 115 (16) ◽  
pp. 3185-3195 ◽  
Author(s):  
Mirle Schemionek ◽  
Christian Elling ◽  
Ulrich Steidl ◽  
Nicole Bäumer ◽  
Ashley Hamilton ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Multipotent Progenitor Cells

Abstract In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.

scholarly journals Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4057-4067 ◽  
Author(s):  
TD Randall ◽  
FE Lund ◽  
MC Howard ◽  
IL Weissman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Mouse Strains ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Adult Bone

Using a monoclonal antibody to murine CD38, we showed that a population of adult bone marrow cells that expressed the markers Sca-1 and c-kit but lacked the lineage markers Mac-1, GR-1, B220, IgM, CD3, CD4, CD8 and CD5 could be subdivided by the expression of CD38. We showed that CD38high c-kit+ Sca-1+, linlow/-cells sorted from adult bone marrow cultured with interleukin-3 (IL-3), IL-6, and kit-L produced much larger colonies in liquid culture at a greater frequency than their CD38low/- counterparts. In addition, we found that CD36low/ - cells contained most of the day-12 colony-forming units-spleen (CFU-S) but were not long-term reconstituting cells, whereas the population that expressed higher levels of CD38 contained few, but significant, day-12 CFU-S and virtually all the long-term reconstituting stem cells. Interestingly, the CD38high Sca-1+ c-kit+ linlow/- cells isolated from day-E14.5 fetal liver were also found to be long-term reconstituting stem cells. This is in striking contrast to human hematopoietic progenitors in which the most primitive hematopoietic cells from fetal tissues lack the expression of CD38. Furthermore, because antibodies to CD38 could functionally replace antibodies to Thy-1.1 in a stem cell purification procedure, the use of anti-CD38 may be more generally applicable to the purification of hematopoietic stem cells from mouse strains that do not express the Thy-1.1 allele.

scholarly journals Diabetes impairs the interactions between long-term hematopoietic stem cells and osteopontin-positive cells in the endosteal niche of mouse bone marrow

2013 ◽  
Vol 305 (7) ◽  
pp. C693-C703 ◽  
Author(s):  
Hironori Chiba ◽  
Koji Ataka ◽  
Kousuke Iba ◽  
Kanna Nagaishi ◽  
Toshihiko Yamashita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mouse Bone Marrow ◽  
Diabetic Mice ◽  
Hematopoietic Stem ◽  
Coculture System ◽  
Endosteal Niche

Hematopoietic stem cells (HSCs) are maintained, and their division/proliferation and quiescence are regulated in the microenvironments, niches, in the bone marrow. Although diabetes is known to induce abnormalities in HSC mobilization and proliferation through chemokine and chemokine receptors, little is known about the interaction between long-term HSCs (LT-HSCs) and osteopontin-positive (OPN) cells in endosteal niche. To examine this interaction, LT-HSCs and OPN cells were isolated from streptozotocin-induced diabetic and nondiabetic mice. In diabetic mice, we observed a reduction in the number of LT-HSCs and OPN cells and impaired expression of Tie2, β-catenin, and N-cadherin on LT-HSCs and β1-integrin, β-catenin, angiopoietin-1, and CXCL12 on OPN cells. In an in vitro coculture system, LT-HSCs isolated from nondiabetic mice exposed to diabetic OPN cells showed abnormal mRNA expression levels of Tie2 and N-cadherin. Conversely, in LT-HSCs derived from diabetic mice exposed to nondiabetic OPN cells, the decreased mRNA expressions of Tie2, β-catenin, and N-cadherin were restored to normal levels. The effects of diabetic or nondiabetic OPN cells on LT-HSCs shown in this coculture system were confirmed by the coinjection of LT-HSCs and OPN cells into bone marrow of irradiated nondiabetic mice. Our results provide new insight into the treatment of diabetes-induced LT-HSC abnormalities and suggest that the replacement of OPN cells may represent a novel treatment strategy.

scholarly journals Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1837-1846 ◽  
Author(s):  
Rashmi Gupta ◽  
Simon Karpatkin ◽  
Ross S. Basch
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Mouse Bone Marrow ◽  
Cell Renewal ◽  
Hematopoietic Stem ◽  
Stem Cell Renewal

Culturing mouse bone marrow in the presence of catalase dramatically alters hematopoiesis. Granulocyte output is initially increased 4- to 5-fold. This increase is transient and granulocyte production declines as immature (Sca-1+/LIN-) cells accumulate. One third of these immature cells have a phenotype (Sca-1+/c-Kit+) characteristic of hematopoietic stem cells. At 2 to 3 weeks there are greater than 200-fold more Sca-1+/c-Kit+/LIN- cells in treated cultures than in controls. This population contains functional stem cells with both short-term and long-term bone marrow repopulating activity. In addition to myeloid progenitors, this Sca-1+/LIN- population contains a large number of cells that express CD31 and CD34 and have an active Tie-2 promoter, indicating that they are in the endothelial lineage. After 3 to 4 weeks hematopoiesis in treated cultures wanes but if catalase is removed, hematopoiesis resumes. After 7 to 10 days the cultures are indistinguishable from untreated controls. Thus, protected from H2O2, hematopoietic progenitors multiply and become quiescent. This sequence resembles in vivo development in normal marrow. These results make it clear that peroxide-sensitive regulatory mechanisms play an important role in controlling hematopoiesis ex vivo and presumably in vivo as well. They also indicate that manipulation of the peroxide levels can be used to enhance the growth of hematopoietic stem cells in culture.

Superior Impact of p18INK4c over p27kip1 on Long-Term Repopulating Ability of Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 796-796
Author(s):  
Hui Yu ◽  
Hongmei Shen ◽  
Xianmin Song ◽  
Paulina Huang ◽  
Tao Cheng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Impact

Abstract The G1-phase is a critical window during the cell cycle in which stem cell self-renewal may be balanced with differentiation and apoptosis. Increasing evidence suggests that the cyclin-dependent kinase inhibitors (CKIs) such as p21Cip1/Waf1, p27kip1, p16INK4A, and p18INK4C (p21, p27, p16 and p18 hereafter) are involved in stem cell self-renewal, as largely demonstrated in murine hematopoietic stem cells (HSCs). For example, we have recently demonstrated a significant increase of HSC self-renewal in the absence of p18 (Yuan et al, Nature Cell Biology 2004). But the actual roles of these CKIs in HSCs appear to be distinct as p21 and p18 have opposite effects (Yu H et al, ASH 2004) whereas p16 has a limited effect (Stepanova et al, Blood 2005) on HSC exhaustion after serial bone marrow transfer. Like p18, however, p27 was recently reported to also inhibit HSC self-renewal due to the fact that the competitive repopulating units (CRUs) were increased in p27−/− mouse bone marrow (Walkley et al, Nature Cell Biology 2005) in contrast to the results in a previous report (Cheng T et al, Nature Medicine 2000). To further gauge the impact of p18 versus p27 on the long-term repopulating ability (LTRA) of HSCs, we have generated different congenic strains (CD45.1 and CD45.2) of p18−/− or p27−/− mice in the C57BL/6 background, allowing us to compare them with the competitive repopulation model in the same genetic background. The direct comparison of LTRA between p18−/− and p27−/− HSCs was assessed with the competitive bone marrow transplantation assay in which equal numbers of p18−/− (CD45.2) and p27−/− cells (CD45.1) were co-transplanted. Interestingly, the p18−/− genotype gradually dominated the p27−/− genotype in multiple hematopoietic lineages and p18−/− HSCs showed 4-5 times more LTRA than p27−/− HSCs 12 months after cBMT. Further self-renewal potential of HSCs was examined with secondary transplantation in which primarily transplanted p18−/− or p27−/− cells were equally mixed with wild-type unmanipulated cells. Notably, while the p18−/− cells continued to outcompete the wild-type cells as we previously observed, the p27−/− cells did not behave so in the secondary recipients. Based on the flow cytometric measurement and bone marrow cellularity, we estimated that transplanted p18−/− HSCs (defined with the CD34−LKS immunophenotype) had undergone a 230-fold expansion, while transplanted p27−/− and wild-type HSCs had only achieved a 6.6- and 2.4-fold expansion in the secondary recipients respectively. We further calculated the yield of bone marrow nucleated cells (BMNCs) per HSC. There were approximately 44 x 103, 20.6 x 103, and 15 x 103 BMNCs generated per CD34−LKS cell in p18−/−, p27−/− and wild-type transplanted recipients respectively. Therefore, the dramatic expansion of p18−/− HSCs in the hosts was not accompanied by decreased function per stem cell. Our current study demonstrates that hematopoietic engraftment in the absence of p18 is more advantageous than that in the absence of p27, perhaps due to a more specific role of p18 on self-renewal of the long-term repopulating HSCs.

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