scholarly journals Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 1779-1787 ◽  
Author(s):  
Claudia R. Ball ◽  
Ingo H. Pilz ◽  
Manfred Schmidt ◽  
Sylvia Fessler ◽  
David A. Williams ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Cell Clones ◽  
Selection For ◽  
The Impact ◽  
Reduced Intensity ◽  
Selection Of

AbstractEfficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However, a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore, understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)–expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K–expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly, not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection, thus underscoring the potential value of MGMT P140K selection for clinical gene therapy.

Efficient MGMTP140K-Mediated In Vivo Selection and Chemoprotection of Long-Term Repopulating Cells in Nonhuman Primates Following Reduced Intensity Conditioning.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3572-3572
Author(s):  
Brian C Beard ◽  
Grant D Trobridge ◽  
Jeannine S McCune ◽  
Hans-Peter Kiem
Keyword(s):  
Nonhuman Primates ◽  
Conditioning Regimen ◽  
Reduced Intensity Conditioning ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Reduced Intensity Conditioning Regimen ◽  
Hematopoietic Toxicity ◽  
Reduced Intensity

Abstract Abstract 3572 Poster Board III-509 Strategies using gene-modified hematopoietic stem cells to treat various severe hematopoietic diseases, including but not limited to hemoglobinopathies, will likely require high levels of gene marking. Here we have established efficient and stable in vivo selection in nonhuman primates using methylguanine methyltransferase (MGMTP140K). In the macaque (Macaca nemestrina) we were able to increase pre-chemotherapy lentiviral gene marking levels of 11.3% in granulocytes and 15.3% in lymphocytes to a post-chemotherapy gene marking level of 76.9% in granulocytes and 49.0% in lymphocytes. Furthermore, stable increases in gene marking were also observed in red blood cells (RBCs) and platelets (PLTs) with a pre-chemotherapy gene marking level of 5.6% and 6.7%, respectively, and a post-chemotherapy gene marking level of 15.2% and 64.0%, respectively. Importantly, the chemotherapy regimen was well tolerated, and engraftment was polyclonal as determined by analyzing long-term repopulating clones by LAM-PCR. In order to minimize extra-hematopoietic toxicity we have began to test a more clinically applicable conditioning regimen in the macaque model. This reduced intensity conditioning regimen should allow treatment of patients with severe hematopoietic or infectious diseases, who may not tolerate a high dose conditioning regimen. We tested targeted busulfan for conditioning to provide sufficient myelosuppression and to facilitate engraftment of chemoprotected hematopoietic stem cells while minimizing extra-hematopoietic toxicity. Following conditioning with busulfan (4 mg/kg/day for 2 days) and infusion of gene modified cells (∼1.7 × 107 CD34-selected cells/kg), there was moderate cytopenia with ANC <500/mL for 7 days and thrombocytopenia with a nadir of 18,000/mL. Following stable hematopoietic recovery, we observed gene marking, determined by RT-PCR, in total white blood cells as a provirus copy number of 0.04 (∼4% gene marking) that, following a single cycle of O6BG (x2) and BCNU, rose to 0.16 (∼16% gene marking). Currently, gene marking has been stable for more than 9 months following chemotherapy. The treatment was well tolerated with only transient elevated liver enzymes following O6BG/BCNU treatment and no additional extra-hematopoietic toxicity has been observed. Clonality studies before and after in vivo selection is underway using a combination of LAM-PCR and a modified whole genome pyrosequencing approach. In summary, we have attained efficient and stable in vivo selection of long-term repopulating cells in nonhuman primates, and have extended this approach to use a reduced intensity conditioning regimen that should be well tolerated in patients with many hematopoietic diseases. Disclosures: No relevant conflicts of interest to declare.

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 ◽  
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.

In Vivo Selection and Long-Term Engraftment Of Hematopoietic Stem Cells Generated Via Vascular Niche Induction Of Nonhuman Primate Induced Pluripotent Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 466-466
Author(s):  
Jennifer L Gori ◽  
Jason M Butler ◽  
Devikha Chandrasekaran ◽  
Brian C Beard ◽  
Daniel J Nolan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Nonhuman Primate ◽  
Fold Increase ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P<0.05). On average, 15% CD34+ and 37% CD45+ cells were detected in the bone marrow of mice transplanted with E4ORF1+EC-expanded P140K-MniPSC HSCs, which is significantly higher than levels detected in the other cohorts (Table 1). CD45+ cells in the marrow were predominantly myeloid but lymphoid subsets were also present (10-25% CD3+ cells). Remarkably, the level of gene marking in CFCs and number of gene marked CFCs from mouse bone marrow was substantially higher for mice transplanted with E4ORF1+EC expanded compared to cytokine expanded P140K-MniPSC-HSCs (Table 1). Finally, to confirm engraftment of authentic HSCs, secondary transplants were established. Although engraftment was achieved in all secondary transplanted cohorts, the level of nonhuman primate cells detected was significantly higher in animals transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs. Significantly more lymphocytes (CD45+CD3+ and CD45+CD56+) and monocytes (CD45+CD14+) were detected in the blood of these secondary transplant recipients. These findings confirm generation of bona fide HSCs derived from nonhuman primate iPSCs and demonstrate that O6BG/BCNU chemotherapy supports in vivo selection of P140K-MniPSC-HSCs generated by co-culture with the E4ORF1+EC vascular platform. Our studies mark a significant advance toward clinical translation of PSC-based blood therapeutics and the development of a nonhuman primate preclinical model. Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0 Disclosures: Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.

scholarly journals Therapeutic in vivo selection of thymic-derived natural T regulatory cells following non-myeloablative hematopoietic stem cell transplant for IPEX

2011 ◽  
Vol 141 (2) ◽  
pp. 169-176 ◽  
Author(s):  
Kimberly A. Kasow ◽  
Vanessa M. Morales-Tirado ◽  
David Wichlan ◽  
Sheila A. Shurtleff ◽  
Allistair Abraham ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplant ◽  
T Regulatory Cells ◽  
Stem Cell Transplant ◽  
Cell Transplant ◽  
Regulatory Cells ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

MGMT (P140K) Allows for Efficient and Sustained In Vivo Selection in Non-Human Primates.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3270-3270
Author(s):  
Brian C. Beard ◽  
Kate Beebe ◽  
Julia Piasecki ◽  
Christina Gooch ◽  
David Dickerson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Negative Selection ◽  
Chemotherapy Treatment ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Elevated Liver Enzymes ◽  
Low Levels ◽  
Selection Of

Abstract In vivo selection strategies that convey a survival advantage to genetically modified cells carrying mutant forms of methylguanine methyltransferase (MGMT-P140K) have the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. For some applications such as genetic diseases or anti-HIV strategies, in vivo selection may be required to increase initially low levels of gene-modified cells while for malignant diseases hematopoietic stem cell (HSC) chemo-protection may be necessary during chemotherapy dose escalation. Thus we have explored the use of gammaretrovirally expressed MGMT(P140K) mutant in three baboons. Animals received CD34−enriched cells transduced with a GALV-pseudotyped retroviral vector expressing a bicistronic message containing P140K and GFP. Two of the animals were part of a competitive repopulation assay in which one half of the cells were gene-modified with a GALV-pseudotyped vector expressing only YFP. After stable engraftment all three baboons were treated with various regimens of O6-benzylguanine (O6BG) and temozolomide (TMZ) or BCNU. Following treatment with O6BG/TMZ the selection was transient for ‘protected’ cells gene-modified with MGMT(P140K)-GFP, and the expected negative selection of ‘unprotected’ gene-modified cells (YFP transgene alone) was subtle. Conversely, positive selection of MGMT(P140K)-GFP gene-modified cells and negative selection of YFP gene-modified cells was dramatic and sustained following treatment with only a single dose of O6BG/BCNU. The increase in gene-marking (up to ~85%) is stable following selection out to 22 months. Importantly, selection of hematopoietic cells was polyclonal and no evidence of insertional mutagenesis has been detected. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. We suspect that the delivery/absorption of TMZ in non-human primates is a contributing factor to transient selection because in animals with low levels (<1%) of MGMT(P140K) gene-modified cells no pronounced or sustained drop in white blood cell or platelet counts was observed following O6BG/TMZ. This is the case even up to TMZ dose levels of 700 mg/m2 that is above dose limiting toxicity in humans. In summary, MGMT selection is efficient and well tolerated in monkeys and we believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy. Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow). Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow).

Successful Gene Therapy of Murine Paroxysmal Nocturnal Hemoglobinuria (PNH) Using Mgmt-Mediated In Vivo Selection of Genetically Corrected, Drug-Resistant Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 173-173
Author(s):  
François Moreau-Gaudry ◽  
Bing Han ◽  
Emmanuel Richard ◽  
Ike E. Pantazopoulos ◽  
Hubert de Verneuil ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Murine Model ◽  
Blood Cells ◽  
Alkylating Agents ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selective Growth Advantage ◽  
Selection Of

Abstract PNH is an acquired disorder of the hematopoietic stem cell (HSC) caused by a somatic mutation in the PIGA gene. Blood cells carrying the mutation are deficient in all glycosyl phosphatidylinositol-anchored proteins (GPI-APs). Long-term restoration of GPI-AP has been obtained in vitro using lentiviral vectors stably expressing PIGA in human HSCs. However, gene therapy for PNH might be problematic because of the lack of a growth advantage of phenotypically corrected cells. We hypothesized that providing corrected cells with a selective growth advantage might overcome this possible limitation. We chose to test this in a murine model of PNH that has GPI-AP deficient blood cells because of a somatic Piga gene mutation targeted to the HSCs (LF mice). We designed a novel bicistronic lentiviral vector (MMIP) expressing the human PIGA cDNA and the human alkylating drug resistance mutant O6-methylguanine DNA methyltransferase (MGMT G156A) under the control of the MND promoter. MGMT G156A confers protection against alkylating agents. Eight LF donor mice were injected with 5-FU. Bone marrow (BM) was harvested 5 days later for MMIP transduction performed twice at MOI 100. Transduced and mock transduced BM cells were injected into 15 lethally irradiated C57Bl/6 recipient mice. One month after BM transplantation (BMT) 8 MMIP-transduced mice were treated with the alkylating agents BCNU and O6-benzylguanine (BG) (5mg/kg and 30mg/kg). BCNU /BC treatment was repeated twice, each one month apart. Six months after BMT the percentage of donor engraftment was 75.1 ± 25% for granulocytes (G) and 88.4 ± 26% for lymphocytes (L) in the control group (n=3). Donor chimerism in the MMIP-transduced group not treated with BCNU/BG was 84.6 ± 25 and 87.8 ± 5 % (n=4). The highest donor chimerism was obtained in mice treated with BCNU/BG, 98.9 ± 0.4 and 98.6 ± 0.5 % (n=8). Next we determined the proportion of donor blood cells with a restored expression of GPI-linked proteins. Only a small proportion of GPI-AP expressing cells was found in mice receiving the mock transduced BM six months after BMT, 22±37% GPI-AP expressing RBCs; 3.1± 2% GPI-AP expressing G, and 0.5 ± 0.2% GPI-AP expressing L. In contrast, mice receiving the MMIP-transduced BM cells but not treated with BCNU/BG showed a higher level of GPI-AP+ cells: 87.8±16% for RBCs, 70.2 ±23% for G and 56.3 ± 13% for L (n=4). However, mice receiving the MMIP-transduced BM and BG/BCNU treatment, achieved an almost complete restoration of GPI-AP on peripheral blood cells: 98±3% of RBCs, 96.4 ±4.7% of G and 89.6 ± 13% of L (n=8). Findings in BM, in methylcellulose progenitor assays, and in secondary spleen colony assays confirmed that restoration of GPI-AP and BCNU/BG selection had occurred at the HSC level. This is the first report of long-term restoration of GPI-APs expression in vivo using a murine model for PNH. Our results demonstrate that BCNU/BG treatment is well tolerated (100% survival) leading to a strong in vivo selection of HSCs with restored expression GPI-AP. The selective growth advantage of converted HSCs after dual gene therapy followed by BCNU/BG treatment allows us to achieve and possibly maintain a high level of hematopoiesis with restored GPI-AP expression. The selection for the corrected HSCs might allow them to overcome a possible growth disadvantage in the competition between PNH and normal HSCs.

FoxO Are Critical Mediators of Hematopoietic Stem Cell Resistance to Physiologic Oxidative Stress.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 439-439
Author(s):  
Zuzana Tothova ◽  
Ramya Kollipara ◽  
Brian J. Huntly ◽  
Benjamin H. Lee ◽  
Diego H. Castrillon ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Dynamic Balance ◽  
Normal Numbers ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Hematopoietic development and long-term homeostasis are tightly regulated by a dynamic balance of stem cell self-renewal and differentiation. To understand the role of the FoxO family of transcription factors in these processes, we studied the impact of somatic deletion of all FoxO genes in the adult hematopoietic system of mice engineered with the interferon-inducible Mx-Cre transgene and various conditional FoxO alleles: FoxO1L/L; FoxO3 L/L; and/or FoxO4 L/L. Cre-mediated excision of FoxO1, FoxO3 and FoxO4 (FoxO1/O3/O4L/L), resulted in myeloid lineage expansion, lymphoid developmental abnormalities and a marked decrease of the lineage-negative, Sca-1+, c-Kit+ (LSK) compartment, containing the hematopoietic stem cell (HSC) population, but normal numbers of myeloid progenitors. Furthermore, FoxO1/O3/O4L/L null bone marrow cells were defective in competitive and noncompetitive repopulation assays. This FoxO deficient defect correlated with enhanced cell cycling and increased apoptosis of HSC cells. Notably, there was an HSC-restricted elevation in the level of reactive oxygen species (ROS) that was not observed in committed progenitors, and was associated with decreased catalase and MnSOD expression. Furthermore, in vivo treatment with the anti-oxidative agent N-acetyl-cysteine (NAC) resulted in complete reversion of the FoxO deficient HSC phenotype. Taken together, these results demonstrate that, in the HSC compartment, FoxO proteins are mediators of quiescence and enhanced survival and play essential regulatory roles in the response to physiologic oxidative stress, a function that may contribute to the long-term regenerative potential of the hematopoietic stem cell compartment.

In Vivo Lentiviral Marking Demonstrates Long-Term Myeloid and Lymphoid Lineage Contribution of Individual Hematopoietic Stem Cell Clones to Murine Steady-State Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 813-813
Author(s):  
Oksana Zavidij ◽  
Claudia R Ball ◽  
Sylvia Fessler ◽  
Daniela Belle ◽  
Manfred Schmidt ◽  
...  
Keyword(s):  
Stem Cell ◽  
Steady State ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Hematopoietic Stem ◽  
Cell Clones ◽  
Stem And Progenitor Cells ◽  
One Year

Abstract Abstract 813 Most of the knowledge to date on the in vivo blood forming activity of individual hematopoietic stem and progenitor cells was gained in transplantation experiments of defined cell populations into syngeneic or xenogeneic murine hosts. Consequently, stem and progenitor cells are solely defined by their role in post-transplant reconstitution and very little is known on their clonal activity in steady-state hematopoiesis. To gain new insights into the clonal activity of stem and progenitor cells under steady-state conditions we used a genetic in vivo lentiviral marking strategy and subsequently monitored the clonal activity of marked hematopoietic cells for up to one year by highly sensitive integration site amplification using LAM-PCR. Highly concentrated GFP-expressing lentiviral vectors (LV) were injected intravenously (IV, n=10) or intrafemorally (IF, n=15) into GFP-tolerant B6.Cg-Tg (Krt1-15-EGFP) 2Cot/J (Krt15) mice to directly mark hematopoietic stem and progenitor cells. 5 mice from each of the two cohorts were treated with 5-Fluorouracil (5-FU, 150 mg/kg) to mobilize hematopoietic stem cells prior to LV-marking. The clonality of the transduced myelopoiesis and lymphopoiesis was analyzed by LAM-PCR. A small proportion of all peripheral blood cells in LV-injected mice consistently expressed GFP for up to one year (5-100 GFP+ cells per 20000 PB cells analyzed). Pre-treatment with 5-FU did not affect the percentage or lineage distribution of marked blood cells even when the vector was injected intravenously. Even though the initial percentage of marked cells was similar after IV and IF vector injection (p>0.05) the marking kinetics were different. Whereas the percentage of GFP expressing cells in PB of IF-marked mice remained stable over the whole observation period for up to 1 year, a 2-fold decline of the percentage of marked cells was detected two weeks after IV-marking indicating that predominantly short-lived more mature cells were transduced after IV vector injection. LAM-PCR analyses of sorted cell lineages showed that multiple clones contributed to the marked myeloid and lymphoid long-term hematopoiesis after IF-injection. In summary, our data demonstrate stable marking of steady-state hematopoiesis for up to one year. Our results demonstrate that remarkably stable stem cell clones with myeloid and lymphoid differentiation potential contribute to murine steady-state long-term hematopoiesis. In vivo marking will further allow to directly address the response of individual stem cell clones to hematopoietic stress including chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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