scholarly journals A cell surface marker gene transferred with a retroviral vector into CD34+ cord blood cells is expressed by their T-cell progeny in the SCID- hu thymus

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 107-113 ◽  
Author(s):  
C Champseix ◽  
V Marechal ◽  
I Khazaal ◽  
O Schwartz ◽  
S Fournier ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cell ◽  
Cell Surface ◽  
Cord Blood ◽  
Blood Cells ◽  
Cell Lineage ◽  
Surface Marker ◽  
Cd34 Cells ◽  
A Cell ◽  
Cord Blood Cells

Gene transduction into immature hematopoietic cells collected at birth from the umbilical cord could be useful for the treatment of genetic or acquired disorders of the hematopoietic system diagnosed during pregnancy. The SCID-hu mouse is a convenient model to investigate T- cell lineage gene therapy, since it allows replication of human intrathymic T-cell development. CD34+ cells isolated from cord blood were cocultured with CRIP MFG-murine CD2 (mCD2) cells that produce recombinant retroviruses encoding the mCD2 antigen, a cell surface marker easily detectable by flow cytometry. After 3 and 4 days in coculture, a mean of 19% and 39% human hematopoietic cells, respectively, expressed the mCD2 antigen. CD34+ cells cocultured for 4 days were used to reconstitute human fetal thymus implanted in SCID mice. Five to 10 weeks later, the mCD2 antigen was detected on approximately 10% of human thymocytes repopulating the thymic grafts in four of nine SCID mouse chimeras. Vector genomes were detected in graft cell DNA by Southern blot. Analysis of vector integration indicated that positive cells were of polyclonal origin in three animals and predominantly monoclonal in the other one. Our data show that foreign genes can be transduced into CD34+ cord blood cells endowed with T-cell differentiation potential, and suggest strategies for T-cell lineage gene therapy in the neonate.

scholarly journals A cell surface marker gene transferred with a retroviral vector into CD34+ cord blood cells is expressed by their T-cell progeny in the SCID- hu thymus

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 107-113 ◽  
Author(s):  
C Champseix ◽  
V Marechal ◽  
I Khazaal ◽  
O Schwartz ◽  
S Fournier ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cell ◽  
Cell Surface ◽  
Cord Blood ◽  
Blood Cells ◽  
Cell Lineage ◽  
Surface Marker ◽  
Cd34 Cells ◽  
A Cell ◽  
Cord Blood Cells

Abstract Gene transduction into immature hematopoietic cells collected at birth from the umbilical cord could be useful for the treatment of genetic or acquired disorders of the hematopoietic system diagnosed during pregnancy. The SCID-hu mouse is a convenient model to investigate T- cell lineage gene therapy, since it allows replication of human intrathymic T-cell development. CD34+ cells isolated from cord blood were cocultured with CRIP MFG-murine CD2 (mCD2) cells that produce recombinant retroviruses encoding the mCD2 antigen, a cell surface marker easily detectable by flow cytometry. After 3 and 4 days in coculture, a mean of 19% and 39% human hematopoietic cells, respectively, expressed the mCD2 antigen. CD34+ cells cocultured for 4 days were used to reconstitute human fetal thymus implanted in SCID mice. Five to 10 weeks later, the mCD2 antigen was detected on approximately 10% of human thymocytes repopulating the thymic grafts in four of nine SCID mouse chimeras. Vector genomes were detected in graft cell DNA by Southern blot. Analysis of vector integration indicated that positive cells were of polyclonal origin in three animals and predominantly monoclonal in the other one. Our data show that foreign genes can be transduced into CD34+ cord blood cells endowed with T-cell differentiation potential, and suggest strategies for T-cell lineage gene therapy in the neonate.

The Acetylation of AML1-ETO Is Required for Leukemogenesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1588-1588
Author(s):  
Lan Wang ◽  
Alexander Gural ◽  
Fabiana Perna ◽  
Xiaojian Sun ◽  
Xinyang Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cord Blood ◽  
Blood Cells ◽  
Target Genes ◽  
Biological Effects ◽  
Self Renewal ◽  
Mouse Leukemia ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cord Blood Cells

Abstract Abstract 1588 Transcription factors and histones are similarly modified through acetylation, phosphorylation, ubiquitination and methylation, which impact on the transcriptional regulation of gene expression and various biological processes in normal and malignant hematopoiesis. The t(8;21) associated AML1-ETO fusion protein is found in 40% of the FAB M2 subtype of acute myeloid leukemia, but how the post-translational modification of AML1-ETO affects its leukemogenicity is largely unknown. Here we show that AML1-ETO directly interacts with the lysine acetyltransferase, p300, via the region containing NHR1 domain and that p300 can acetylate two lysine residues in AML1-ETO and AML1-ETO (exon 9a) in human and mouse leukemia cells. To understand the biological effects of AML1-ETO acetylation, we used human CD34+ cord blood cells as a preleukemia model. The maintenance of CD34+ cells by the acetylation defective form of AML1-ETO was 5 fold less than with AML1-ETO (p<0.01) in the liquid culture assay, and unlike the effect of AML1-ETO, the number of the cobble stone area forming cells (CAFC) was not increased by the mutant AML1-ETO in CAFC assay. However, the block in erythroid and myeloid differentiation conferred by AML1-ETO was still seen in the AML1-ETO acetylation mutant transduced human CD34+ cells. We then approved the impact of acetylation on leukemogenicity using the AML1-ETO9a (AE9a) mouse leukemia model. Mice receiving AE9a acetylation mutant transduced fetal liver cells have not developed leukemia by Day 250, whereas all the mice receiving AE9a transduced cells died due to leukemia before Day 160, with a mean survival time of 109 days (p<0.001). These results suggest that the acetylation of AML1-ETO is required not only for its self-renewal promoting effects and but also for the development of acute leukemia. To gain insight into the mechanisms of AML1-ETO acetylation, we performed luciferase assays and found that the AML1-ETO acetylation mutant lost the ability to activate an M-CSFR promoter driven reporter construct. Furthermore, the expression levels of AML1-ETO activated target genes related to self-renewal were not upregulated in AML1-ETO acetylation mutant transduced human CD34+ cells. These results indicated that the acetylation is crucial to AML1-ETO induced transcription activation. We have also been studying the role of the region containing NHR1 domain (245 to 430 aa) in AML1-ETO: deletion of this region abrogated the binding of p300 to AML1-ETO and led to loss of AML1-ETO lysine acetylation. Furthermore, loss of the region containing NHR1 domain abrogated the self-renewal properties of AML1-ETO and the activation of AML1-ETO target genes in human CD34+ cord blood cells, without affecting its differentiation-blocking activity or its ability to repress gene expression. Given the importance of the acetylation of AML1-ETO in its biological effects, we inhibited p300 function, chemically and using RNA interference; this blocked the transcriptional activation of AML1-ETO target genes, and inhibited the growth of AML1-ETO expressing AML cells in both pre-leukemic and leukemia models. All together, we have found that the acetylation of AML1-ETO via p300 is indispensable for its leukemia-promoting activity and for its ability to activate gene expression. Our work suggests that inhibition of p300 function may represent an important new anti-leukemia strategy that targets self-renewing, leukemia-initiating cells. Disclosures: No relevant conflicts of interest to declare.

In Vitro Assessment of Tolerance and Rejection Occurring After Co-Transplantation of Allogeneic Umbilical Cord Blood and Haploidentical CD34+ Cells as Treatment for Severe Aplastic Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2352-2352
Author(s):  
Nicole J. Gormley ◽  
Aleah Smith ◽  
Maria Berg ◽  
Lisa Cook ◽  
Catalina Ramos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cord Blood Transplantation ◽  
Neutrophil Recovery ◽  
Post Transplant ◽  
Blood Transplantation ◽  
Cd34 Cells ◽  
Cord Blood Cells

Abstract Abstract 2352 Introduction/Methods: The administration of highly purified haploidentical peripheral blood CD34+ cells combined with an unrelated cord blood transplant results in earlier neutrophil engraftment than is typically seen with a cord blood transplant alone. Chimerism data from pilot trials evaluating this strategy have reported 3 phases of engraftment: 1) early myeloid engraftment from transplanted haplo-CD34+ cells followed by 2) cord blood engraftment resulting in dual chimerism and 3) the subsequent disappearance of haploidentical donor cells with resultant full donor cord chimerism. The mechanism accounting for the disappearance of haploidentical cells has not been defined. Here the clinical results and an in vitro assessment of alloreactivity in three patients that underwent combined haploidentical CD34+ cell and cord blood transplantation for severe aplastic anemia (SAA) are described. The conditioning regimen consisted of cyclophosphamide (60mg/kg/day on days -7 and -6), fludarabine (25mg/m2/day on days -5 to -1), horse ATG (40mg/kg/day on days -5 to -2), and total body irradiation (200cGy on day -1). GVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil. PCR of STRs was used to assess chimerism in T-cell and myeloid lineages and mixed lymphocyte reaction assays(MLR) were performed on peripheral blood samples collected at different time-points post-transplant to assess for alloreactivity against the recipient, the haploidentical donor, or the cord unit. Stimulator cord blood cells for the MLR were obtained from residual cord blood cells remaining in the infusion bag after patient administration and expanded in vitro using anti-CD28/CD3 Dynabeads. Results: Prior to transplantation, all three pts had transfusion dependent SAA associated with severe neutropenia that was refractory to conventional immunosuppressive therapy. Pt 1 had an early transient myeloid recovery (ANC 400 on day+11) from the haploidentical donor followed by engraftment of the cord unit (Cord ANC > 500) on day 21. The patient is currently 2 years post transplant and has 100% cord blood chimerism and is transfusion independent. An MLR assay performed when donor T-cell chimerism was 100% cord, showed evidence for rejection of the haploid cells by cord blood T-cells, with the MLR response to haploidentical donor cells being seven fold higher than the response to fully HLA-mismatched 3rd party cells. In pt 2, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing no evidence for cord engraftment in either myeloid or T-cell lineages at any point post-transplant. The patient is currently 15 months post transplant and is transfusion independent with normal blood counts and sustained “split” chimerism (T-cells recipient in origin with myeloid cells being 100% haploidentical donor). MLR assays showed that the recipient was tolerant to the haploid donor, with no statistically significant difference in the alloreactive response to the haploid donor compared to self. In pt 3, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing split chimerism (T-cell chimerism >90% cord and myeloid chimerism 88–100% haploid donor in origin). MLR assays again showed evidence of rejection of the haploid cells by cord blood T-cells, with a trend towards greater alloreactivity against the haploid donor compared to an HLA mismatched 3rd party on post-transplant day +63. Conclusions: Combined haploidentical CD34+ cell and unrelated cord blood transplantation following highly immunosuppressive conditioning represents a viable treatment option for patients with SAA who lack an HLA-matched donor. Using this approach, 2 of 3 pts had cord blood engraftment associated with early neutrophil recovery from the haploidentical donor. In one pt, the cord unit failed to engraft. Remarkably, sustained engraftment from the haploidentical donor in this pt resulted in transfusion independence. MLR appears to be a useful approach to assess the in vitro alloreactivity of this unique stem cell graft source. In the two pts who had cord engraftment, in vitro MLR assessments established that the disappearance of haploid cells occurred as a consequence of rejection of the haploidentical cells by engrafting cord blood T-cells, rather than from non-immunological haploidentical cell graft failure. Disclosures: No relevant conflicts of interest to declare.

Purification of CD34+ Cells Is Essential for Optimal Ex Vivo Expansion of Umbilical Cord Blood Cells

1997 ◽  
Vol 6 (2) ◽  
pp. 145-150 ◽  
Author(s):  
ROBERT A. BRIDDELL ◽  
BRENT P. KERN ◽  
KERRY L. ZILM ◽  
GREGORY B. STONEY ◽  
IAN K. McNIECE
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Blood Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cd34 Cells ◽  
Cord Blood Cells ◽  
Umbilical Cord Blood Cells

Acquisition of CD34 Correlates with Increased Hematopoietic and Self Renewal Activity of CD34−CD133+ Cord Blood Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4143-4143
Author(s):  
Katharina S. Götze ◽  
Matthias Schiemann ◽  
Stefanie Marz ◽  
Christian Peschel ◽  
Robert A.J. Oostendorp
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Division ◽  
Cord Blood ◽  
Blood Cells ◽  
Self Renewal ◽  
Cd34 Cells ◽  
Cord Blood Cells ◽  
Cd34 Expression ◽  
Hematopoietic Activity

Abstract CD34 is a sialomucin expressed on hematopoietic cells, endothelial cells and muscle satellite cells. Within the hematopoietic system, CD34 expression has been associated with very immature progenitor cells as well as hematopoietic stem cells (HSC), and it is widely used to assess stem cell activity in clinical protocols. In the past, HSC activity was thought to be retained exclusively in the subset of cells expressing CD34. This view has been challenged by recent observations in mice in which HSC activity was also found in the CD34-negative fraction. These findings have since been reproduced using human marrow and cord blood cells. However, the exact relationship between CD34+ and CD34− stem cells remains unclear. We investigated the regulation of CD34 expression as dependent on cell division history. To follow cell division, human cord blood cells were labeled with the fluorescent dye CFSE. Lin-CD34−CD133+CFSE+ (CD34−) and CD34+ populations were almost indistinguishable in their ability to produce CAFCweek6 content. After three days of serum-free culture with stem cell factor, Flt3 ligand and thrombopoietin, almost all initially CD34− cells had acquired expression of CD34, including all undivided cells. We found that, in cultures initiated from CD34− cells, virtually all CAFCweek6 were produced from the divided, now CD34+ cells, indicating these cells had self-renewed. In contrast, similar cultures from initially CD34+ cells demonstrated that hematopoietic activity associated with the undivided cell fraction. We did not find any hematopoietic activity in the cell fraction that remained CD34− or the fraction that lost CD34 after division. Analysis of mRNA expression showed that CD34− and CD34+ cells expressed almost equal levels of CD34, AC133, Flt1, Flk1 and Flt4, while CD34− cells expressed significantly lower levels of Tie1 and Tie2 than CD34+ cells. The expression of CD34 message in CD34− cells was explained by our observation that these cells contained intracellular CD34, indicating that they are “primed” to express the antigen on their cell surface. In conclusion, Lin−CD34−CD133+ cells acquire expression of CD34, even in the absence of cell divisions. These CD34− cells self-renew more rapidly in vitro than cells initially expressing CD34, and self-renewal is preceded by acquisition of CD34 antigen.

Cord Blood as a Very Valuable Source of Neonatal Cells but Embryonic-Like Nature Reevaluated

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2897-2897 ◽  
Author(s):  
Anja Buchheiser ◽  
Stefanie Liedtke ◽  
Amelie Pia Houben ◽  
Simon Waclawczyk ◽  
Milaid Stephan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Blood Cells ◽  
Stem Cell Markers ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cord Blood Cells

Abstract Human umbilical cord blood has become a very valuable source for hematopoietic transplantation. Our group was able to show that CB contains non-hematopoietic stem cells, which were called unrestricted somatic stem cells (USSCs) with a multipotent differentiation potential. These cells have the potential to differentiate into different germ layers (Kögler et al. 2004, Kögler et al. 2005, Kögler et al. 2006, Sensken et al. 2007, Greschat et al. 2008, Ghodsizad A et al. 2008, Trapp et al. 2008). Some studies have now reported a presumably embryonic like nature of cord blood cells. However, Nanog and Oct 4 harbours potential pitfalls for data misinterpretation due to pseudogenes and alternative spliced variants (Liedtke et al. 2007, Liedtke et al. 2008). The related data based on the stem cell markers Nanog and Oct4 concerning these results remain questionable. Therefore, we evaluated the embryonic-like nature of MNCs (n=7), USSC (n=7), CD34+ cells (n=7) derived from cord blood, MNCs from peripheral blood (n=3), MNCs (n=7) and MSCs (n=3) from bone marrow. Using RT-PCR, quantitative RT-PCR and immunohistochemistry, we studied the expression of the pluripotency markers Oct4, Nanog, Sox2 as well as the transcription factors Klf4 and cMyc utilized for the induction of pluripotent stem cells from adult human fibroblasts. The expression level of the transcription factors Klf4 and cMyc was nearly equal in all USSC cell lines and BM MSCs. We neither detected expression of Oct4, Nanog and Sox2 in all tested USSC cell lines nor in MNCs and CD34+ cells from cord blood nor in MSCs, MNCs and CD34+ cells from bone marrow. To increase the sensitivity of our method we performed quantitative Oct4 PCRs. This method revealed that USSCs reach the same Oct4 expression level as human dermal fibroblasts. These results are also supported by the inactive status of the telomerase. As a positive control we used the embryonic carcinoma cell line nTERA-2 showing a high expression of Oct4, Nanog and Sox2. In addition we were able to show that the markers SSEA1, SSEA3 and SSEA4 cannot be used as markers of an embryonic-like phenotype. SSEA-1 recognizes the CD15 epitope, SSEA-4 cross-reacts with an adult MSC subpopulation and SSEA3 was always negative applying the correct isotype controls. However, cord blood does not have to contain embryonic like cells, but it contains neonatal cells as USSC expressing Sox17. For hematopoietic stem cells (HSC) it had already been shown that the transcription factor Sox 17 is required to maintain fetal and neonatal HSC and distinguishes their transcriptional regulation from adult HSCs (Kim et al. 2007). Our results indicate that USSCs and cord blood are neonatal cells without expression of typical embryonic stem cell markers. In more than 10.000 unrelated Cord blood transplants performed so far, no tumor formation associated with an Oct4 positive cell/teratoma formation was observed. Therefore the embryonic-like nature of cord blood cells must be reconsidered.

MN1 Overexpression Promotes Proliferation and Self-Renewal of Primitive Normal Human Hematopoietic Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 681-681
Author(s):  
Suzan Imren ◽  
Michael Heuser ◽  
Ling-Yi Chen ◽  
Glen Edin ◽  
Connie J. Eaves ◽  
...  
Keyword(s):  
Cord Blood ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Hematopoietic Cells ◽  
Human Cord Blood ◽  
Genetic Event ◽  
Final Population ◽  
Cd34 Cells ◽  
Large Expansion ◽  
Cord Blood Cells

Abstract Overexpression of MN1 is a negative prognostic factor in patients with acute myeloid leukemia (AML) with normal cytogenetics. We have previously demonstrated that overexpression of MN1 is sufficient as a single genetic event to induce AML in mice with very short latency (Blood110:1639, 2007). To investigate the effects of MN1 on the functional activity of primitive human hematopoietic cells, we transduced CD34+ human cord blood cells with an oncoretrovirus (3 experiments) or lentivirus (3 experiments) encoding GFP ± MN1. Immediately post-transduction primary colony-forming cell (CFC) assays showed no significant differences in the number or type of colonies generated from the MN1 and control-transduced cells. However, the number of CFCs detectable in secondary assays of the MN1-transduced cells was 17-fold higher than from the GFP-transduced cells and this difference was found to increase a further 234-fold as detected by tertiary CFC assays. To assess the effect of MN1 on cells that can be propagated for more prolonged periods on mouse fibroblast feeders engineered to produce human Steel factor, IL-3 and G-CSF, 104 unsorted cord blood cells were plated into such cultures and the number and types of cells present was then determined 8 weeks later. The remarkable potency of MN1 in stimulating the output of primitive cells under these long-term culture (LTC) conditions was evident from the 1700 greater increase in total cell output and 277-fold higher output of CFCs as compared to control cultures and an accompanying large expansion of CD34+ cells (representing up to 18 % of the final population) whereas CD34+ cells were no longer detectable in the control cultures. Further analysis by limit dilution assay (2 experiments) showed that the frequency of cells able to produce CFCs for at least 6 weeks in these stromal based cultures was increased 10-fold (1/7656 control cells vs 1/718 MN1-transduced cells). In addition the CFC output of each of these was enhanced, on average, 6-fold. Remarkably, these effects of MN1 were sustained for a further 16 weeks as shown by the analysis of the cells they produced in secondary and tertiary LTCs. Overall this resulted in a cumulative 6250-fold increase in total cells over 24 weeks, whereas control cells declined to undetectable levels within 16 weeks. Cells harvested from the secondary and tertiary LTCs initiated with MN1-transduced cells also showed a continuing output of 878,967 ± 332,300 and 264,458 ± 120,600 CFCs after 16 and 24 weeks, respectively. This study sets the stage for further investigation of the effect of MN1 on human NOD/SCID/γcnull mouse repopulating cells and the molecular mechanisms by which MN1 blocks the differentiation fate of primitive human hematopoietic cells and how this may be related to its contribution to the genesis of poor prognosis AML.

ETV6-RUNX1 (TEL-AML1) Deregulates the Erythropoietin Receptor (EPOR) and Promotes Cell Survival.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3641-3641
Author(s):  
Veronica Torrano Moya ◽  
Penny Cardus ◽  
Julia Procter ◽  
Mel Greaves ◽  
Anthony Ford
Keyword(s):  
Cell Surface ◽  
Cord Blood ◽  
Cell Survival ◽  
Blood Cells ◽  
Genetic Alterations ◽  
Erythropoietin Receptor ◽  
Luciferase Reporter ◽  
Erythroid Cell ◽  
Epo Receptor ◽  
Cord Blood Cells

Abstract Abstract 3641 ETV6-RUNX1 (TEL-AML1) fusion is usually an initiating and pre-natal event in childhood acute lymphoblastic leukaemia (ALL). Transformation results in the generation of a persistent pre-leukemic clone, which post-natally converts to ALL following the acquisition of necessary secondary genetic alterations. The erythropoietin receptor gene (EPOR) is consistently highly expressed ectopically in TEL-AML1+ ALL but the presence of a functional receptor on the cell surface and its role, if any, in leukemogenesis driven by TEL-AML1 remains to be confirmed. Similarly, there is much debate as to the role played by the cytokine erythropoietin (EPO) and signaling through EPOR in relation to non-erythroid cell survival. Here, we show by using biotinylated erythropoietin (EPO) and flow analysis that the pre-B ALL TEL-AML1+ cell line (REH) appeared to have higher levels of EPO receptor than other non-TEL-AML1 cell lines. Furthermore, when we “blind screened” CD19+ peripheral blood or bone marrow cells from 10 patients with pre-B ALL, we identified 5 patients that showed high expression of ligand-binding EPO receptor on the cell surface, 4 of which were subsequently shown to be TEL-AML1+. We show that the inducible expression of TEL-AML1 in lymphoid BaF3 cells or its constitutive expression in either a murine transgenic model or in normal human cord blood cells is also sufficient to increase expression of EPOR. In order to further assess the direct regulation of EPOR by TEL-AML1 we next performed EMSA and ChIP experiments to confirm occupancy of AML1 consensus binding sites within the EPOR promoter and luciferase reporter assays to confirm up-regulation of EPOR promoter activity in the presence of TEL-AML1. Given the proposed pro-survival properties of EPO on non-erythroid cells, we next asked if the observed increase in expression of the EPOR in cells expressing TEL-AML1 could correlate with an increased cell survival in the presence of EPO. Cell survival experiments including growth curves, Annexin V staining and analysis of anti-apoptotic gene markers revealed that IL3-dependent cells expressing TEL-AML1 showed a prolonged survival in the presence of EPO alone and the consequent activation of anti-apoptotic pathways. The subsequent removal of TEL-AML1 expression in these cells resulted in cell death even in the presence of EPO, further suggesting that this effect is a consequence solely of fusion gene expression. Moreover, the observed survival in the presence of TEL-AML1 and EPO was enhanced by the co-addition of stem cell factor (SCF). Interestingly, SCF associated intracellular tyrosines have been shown to play an essential role in signaling through EPOR-SCFR interactions. In the presence of EPO, signaling via EPOR was confirmed by phosophorylation of JAK2, STAT5 and AKT and the subsequent up-regulation of BCL-XL. Similarly, EPOR functionality was demonstrated in TEL-AML1 positive patient cells by analysis of JAK2 pathway signaling after EPO induction. We propose that such activation lead to the increase in cell survival also observed in patient cells in the presence of EPO - again suggesting a role for enhanced cell survival through the EPO-EPOR axis. In our final model of pre-leukemia, CD34+ cord blood cells were transduced in vitro with a lentivirus capable of expressing both TEL-AML1 and GFP and ‘primed’ for pre-B lineage commitment. GFP (TEL-AML1) positive cord blood cells also showed increased levels of cell surface EPO receptor when compared to empty vector controls. Collectively, these data support the contention that TEL-AML1 directly activates ectopic expression of a functional EPO receptor and provides cell survival signals that may contribute critically to persistence of the pre-malignant clone in patients. Disclosures: No relevant conflicts of interest to declare.

Prenatal Origin of Monosomy 7 in Very Young Children.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2557-2557
Author(s):  
Parinda A. Mehta ◽  
Mark Wunderlich ◽  
Teresa Smolarek ◽  
Stella M. Davies ◽  
Sarah Zimmerman ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Young Children ◽  
Cord Blood ◽  
Blood Cells ◽  
Total Sample ◽  
Monosomy 7 ◽  
Very Young Children ◽  
Cd34 Cells ◽  
Cord Blood Cells

Abstract Abstract 2557 Monosomy 7 is a common chromosomal abnormality in adults with myelodysplasia (MDS) and AML, seen most frequently in older persons. It is believed to be one of a series of stochastically acquired genetic anomalies that accumulate over many years to complete the process of leukemogenesis. MDS in general and monosomy 7 in particular are rarely seen in children, although cases in very young children are reported. Monosomy 7 arising in very young children likely has a different etiology and may contribute differently to leukemogenesis, as the young age of the children does not allow sufficient time for stochastic acquisition of mutation in the same way as in elderly persons. A proportion of young children with monosomy 7 have an associated DNA instability disorder such as Fanconi anemia (FA) or Dyskeratosis Congenita, and this genetic background likely plays a key role in the evolution of malignancy. In a few reported cases, the occurrence of monosomy 7 is familial, and in a smaller number of cases yet, the clone may resolve spontaneously or wax and wane, as the associated pancytopenia waxes and wanes. We report 5 young children with monosomy 7 in whom we have explored the origins of monosomy 7. In case 1, who presented with MDS and monosomy 7 at 2 years of age, a stored cord blood was available for analysis. The sample was flow sorted and FISH for monosomy 7 was performed on different populations. The monosomy 7 clone was present in approximately 10% of the cord blood cells overall, and the same clone was found in the most primitive population, i.e. CD34+CD38- cells, suggesting in utero origin of this abnormal clone in a very early precursor or stem cell population (Table 1). Similar analyses done on BM or PB samples of 4 additional young patients with monosomy 7, with age range of 4 months to 42 months (3 ½ years)(median 22.5 months), also showed the abnormal clone to be present in CD34+ cells, supporting our hypothesis that monosomy 7 arises in early precursors or stem cells (Table 2, below). In fact, we were able to confirm the presence of monosomy 7 in the CD34+CD38- population in one of these additional cases for which sufficient material was available. Interestingly, in one patient (#4) where we had sequential samplings nine months apart, total BM showed a decrease in the percentage of cells with monosomy 7, from 2.6% to 1.1%. However, in sorted CD34+ cells, we detected a nearly 10 fold increase in cells with monosomy 7 (Table 2). Clinical significance of this finding remains to be determined In summary, our preliminary studies provide proof of concept that in children, the abnormal monosomy 7 clone is present in the hematopoietic stem cell compartment, and can arise in utero in the absence of any known genetic susceptibility syndrome. Table 1. Patient 1 (UCB) FACS Analysis: % of Cord Blood Cells % Monosomy 7 by FISH CD3 (T cells) 23.1% 6/66 (9.1%) CD 19 (B cells) 9.1% 1/44 (0.02%) CD 14 (monocytes) 4% 1/8 (12.5%) CD 16 (granulocytes) 21.5% 0/20 (0%) CD34 1% 70% CD34+CD38- (primitive progenitor) NA 124/136 (91.2%) CD34+38+19-7- (myeloid progenitor) NA 8/13 (61.5%) Table 2 Patient # (BM or PB) % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH CD3 (T cells) CD 19 (B cells) CD 14 (monocytes) CD 16 (granulocytes) CD34+38+ or CD34+ ( progenitor) CD34+CD38- (primitive progenitor) 2 BM 27.0 0.4 1.9 10.9 5.9 46.4 11.3 16.9 1.2 77 (34+) NA NA 3 BM 40.3 2.5 6.6 1.7 2.4 94 15.9 92.4 0.24 78.3 (34+) .01 NA 4-1 BM 37.4 0 13.3 0 3.7 5.9 15.0 3.8 3.9 1.3 1.7 2.8 4-2 BM 13.0 2 9.5 4 0.4 0 35.6 0.4 0.2 12.2 (34+) NA NA 5 PB 21.8 1.5 3.4 NA 22.4 100 9.1 100 NA NA NA NA Disclosures: No relevant conflicts of interest to declare.

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