Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.

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Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

Blood ◽

10.1182/blood.v83.4.939.939 ◽

1994 ◽

Vol 83 (4) ◽

pp. 939-948 ◽

Cited By ~ 116

Author(s):

Y Tomita ◽

DH Sachs ◽

M Sykes

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Whole Body ◽

Mixed Chimerism ◽

Hematopoietic Stem ◽

Donor Cells ◽

Donor Type ◽

Low Levels

Abstract We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.

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

Blood ◽

10.1182/blood.v84.1.74.74 ◽

1994 ◽

Vol 84 (1) ◽

pp. 74-83 ◽

Cited By ~ 36

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.

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Host Conditioning With 5-Fluorouracil and kit-Ligand to Provide for Long-Term Bone Marrow Engraftment

Blood ◽

10.1182/blood.v89.7.2376 ◽

1997 ◽

Vol 89 (7) ◽

pp. 2376-2383 ◽

Cited By ~ 25

Author(s):

Ronald van Os ◽

Donald Dawes ◽

John M.K. Mislow ◽

Alice Witsell ◽

Peter M. Mauch

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Mixed Chimerism ◽

Kit Ligand ◽

Donor Bone Marrow ◽

Before And After ◽

Total Body ◽

Marrow Cells

Abstract Administration of kit-ligand (KL) before and after doses of 5-fluorouracil (5-FU) results in marrow failure in mice, presumably because of enhanced KL-induced cycling of stem cells, which makes them more susceptible to the effects of 5-FU. In attempt to capitalize on this effect on stem cells, we studied the ability of KL and 5-FU to allow stable donor engraftment of congenically marked marrow in a C57BL/6 (B6) mouse model. KL was administered subcutaneously at 50 μg/kg, 21 hours and 9 hours before and 3 hours after each of two doses of 5-FU (125 mg/kg) given 7 days apart to B6-recipients. Animals then received three injections of 107 congenic B6-Gpi-1a-donor bone marrow cells at 24, 48, and 72 hours after the second 5-FU dose. A separate group of animals received a single dose of either 1 × 107 or 3 × 107 donor marrow cells 24 hours after the last 5-FU dose. The level of engraftment was measured from Gpi-phenotyping at 1, 3, 6, and 8 months in red blood cells (RBCs) and at 8 months by phenotyping cells from the thymus, spleen, and marrow. Percent donor engraftment in RBCs appeared stable after 6 months. The percent donor engraftment in RBCs at 8 months was significantly higher in KL + 5-FU prepared recipients (33.0 ± 2.7), compared with 5-FU alone (18.5 ± 2.6, P < .0005), or saline controls (17.8 ± 1.7, P < .0001). In an additional experiment, granulocyte colony-stimulating factor (100 μg/dose) was added to a reduced dose of KL (12.5 μg/dose); engraftment was similar to KL alone. At 8 months after transplantation the levels of engraftment in other tissues such as bone marrow, spleen, and thymus correlated well with erythroid engraftment to suggest that multipotent long-term repopulating stem cells had engrafted in these animals. There are concerns for the toxicity of total body irradiation (TBI)- or busulfan-based regimens in young recipients of syngeneic or transduced autologous marrow who are transplanted for correction of genetic disease. In these recipients complete donor engraftment may not be needed. The results with KL and 5-FU are encouraging for the further refinement of non-TBI, nonbusulfan techniques to achieve stable mixed chimerism.

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Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and Thy-1 and is highly enriched for Abelson leukemia virus targets.

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2665 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2665-2671 ◽

Cited By ~ 26

Author(s):

G F Tidmarsh ◽

S Heimfeld ◽

C A Whitlock ◽

I L Weissman ◽

C E Müller-Sieburg

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Leukemia Virus ◽

Cell Activity ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Low Levels ◽

B Lineage ◽

Marrow Cells

A novel stage in early B-lymphocyte differentiation has been identified in normal mouse bone marrow cells. Earlier work had demonstrated that bone marrow cells characterized by low levels of Thy-1 and lack of a panel of lineage markers (Thy-1lo Lin- cells) were highly enriched for pluripotent hematopoietic stem cells. In this paper, we present evidence that another bone marrow population, which expressed low levels of Thy-1 and coexpressed B220, a B-lineage-specific form of the leukocyte common antigen, contained early and potent precursors for B lymphocytes upon in vivo transfer to irradiated hosts. These Thy-1lo B220+ cells, comprising 1 to 2% of bone marrow cells, were enriched for large cells in the mitotic cycle; the population lacked significant pluripotent hematopoietic stem cell activity and myeloid-erythroid progenitors. Most strikingly, Thy-1lo B220+ cells represented a highly enriched population of bone marrow cells that could be targets of Abelson murine leukemia virus transformation. We propose that Thy-1lo B220+ bone marrow cells represent the earliest stage of committed lymphocyte progenitors, intermediate in differentiation between Thy-1lo Lin- pluripotent stem cells and, in the B lineage, Thy-1- B220+ pre-B cells.

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Long-Term Follow-Up of Serially Transplanted Mice Receiving Extended Reduced Intensity Selection of MGMT-P140K Expressing Murine Repopulating Stem Cells Demonstrates Stable Oligo- to Polyclonal Hematopoiesis without Clonal Exhaustion.

Blood ◽

10.1182/blood.v106.11.1286.1286 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1286-1286

Author(s):

Claudia Ball ◽

Manfred Schmidt ◽

Ingo Pilz ◽

Monika Schrempp ◽

Christof von Kalle ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Blood Cells ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Reduced Intensity ◽

Selection Of

Abstract In vivo selection of gene modified hematopoietic stem cells permanently increases the relative proportion of blood cells that carry a therapeutic transgene despite initially low gene transfer efficiency, thereby decreasing the likelihood of insertional mutagenesis and avoiding the need of myeloablative conditioning regimens. P140K Mutant O6-methylguanine-DNA methyltransferase (MGMT) enzyme confers resistance to the combination of the MGMT inhibitor O(6)-benzylguanine (O(6)BG) and nitrosourea drugs such as 1,3-bis-(2 chloroethyl)-1-nitrosourea (BCNU). We have previously shown that reduced intensity and toxicity BCNU/O6-BG selection allows efficient selection of MGMT-P140K expressing oligoclonal murine hematopoiesis. Nevertheless, whether long-term selection and the associated proliferative stress impairs long-term differentiation and proliferation of MGMT-P140K expressing stem cell clones is currently unknown and remains a major concern in the clinical application of MGMT selection. To address this question, serial transplantations of murine MGMT-P140K expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were done. After ex vivo gene transfer of an MGMT/IRES/eGFP encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and primary, secondary and tertiary recipient mice were subsequently treated every four weeks in order to exaggerate potential effects on long-term clonal behaviour. Lineage contribution of the transduced hematopoiesis was monitored by FACS over a total of 14 rounds of selection and clonality by LAM-PCR over a total of 12 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 persisting peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential of transduced cells in 1st, 2nd and 3rd generation animals. LAM PCR analysis of peripheral blood samples revealed stable oligo- to polyclonal hematopoiesis in primary and secondary mice. Evidence for predominant clones or clonal exhaustion was not observed despite up to 12 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice that received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis, indicating extensive self-renewal of transplantable stem cells 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 their differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies for the amplification of a limited number of gene corrected clones in clinical gene therapy.

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

Blood ◽

10.1182/blood.v108.11.3271.3271 ◽

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.

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Cellular basis of graft versus host tolerance in chimeras prepared with total lymphoid irradiation.

Journal of Experimental Medicine ◽

10.1084/jem.152.3.736 ◽

1980 ◽

Vol 152 (3) ◽

pp. 736-741 ◽

Cited By ~ 4

Author(s):

M Gottlieb ◽

S Strober ◽

H S Kaplan

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Suppressor Cells ◽

Third Party ◽

Whole Body ◽

Mixed Chimerism ◽

Clonal Deletion ◽

Donor Type ◽

Host Tolerance ◽

Marrow Cells

BALB/c mice given allogeneic (C57BL/Ka) bone marrow cells after toal lymphoid irradiation become stable chimeras approximately 80% donor-type and 20% host-type cells in the spleen. The chimeras doe not develop graft vs. host disease (GVHD). Purified cells of C57BL/Ka origin from the chimeras mediated GVHD in lightly irradiated C3H (third party), but not in BALB/c (host-strain) mice. Thus graft vs. host tolerance in the chimeras could not be explained by complete immunodeficiency of donor-type cells, serum blocking factors, or suppressor cells of host (BALB/c) origin. Clonal deletion or suppression of lymphocytes reactive with host tissues remain possible explanations. The transfer of donor-type chimeric spleen cells to BALB/c recipients given 500-550 rad whole-body irradiation WBI led to stable mixed chimerism in approximately 50% of recipients. The cells were presumably acting as tolerogens because similarly irradiated BALB/c mice given (BALB/c X C57BL/Ka)F1 spleen or bone marrow cells also became stable mixed chimeras.

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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.

Journal of Experimental Medicine ◽

10.1084/jem.175.1.175 ◽

1992 ◽

Vol 175 (1) ◽

pp. 175-184 ◽

Cited By ~ 250

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.

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

Blood ◽

10.1182/blood-2009-04-215376 ◽

2010 ◽

Vol 115 (16) ◽

pp. 3185-3195 ◽

Cited By ~ 64

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.

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