Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis

Blood ◽  
2009 ◽  
Vol 114 (8) ◽  
pp. 1498-1505 ◽  
Author(s):  
Aleksandra Rizo ◽  
Sandra Olthof ◽  
Lina Han ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Oxygen Species ◽  
Self Renewal ◽  
Human Cd34 ◽  
Bmi1 Expression ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Cord Blood Cd34

Abstract High expression of BMI1 in acute myeloid leukemia (AML) cells is associated with an unfavorable prognosis. Therefore, the effects of down-modulation of BMI1 in normal and leukemic CD34+ AML cells were studied using a lentiviral RNA interference approach. We demonstrate that down-modulation of BMI1 in cord blood CD34+ cells impaired long-term expansion and progenitor-forming capacity, both in cytokine-driven liquid cultures as well as in bone marrow stromal cocultures. In addition, long-term culture-initiating cell frequencies were dramatically decreased upon knockdown of BMI1, indicating an impaired maintenance of stem and progenitor cells. The reduced progenitor and stem cell frequencies were associated with increased expression of p14ARF and p16INK4A and enhanced apoptosis, which coincided with increased levels of intracellular reactive oxygen species and reduced FOXO3A expression. In AML CD34+ cells, down-modulation of BMI1 impaired long-term expansion, whereby self-renewal capacity was lost, as determined by the loss of replating capacity of the cultures. These phenotypes were also associated with increased expression levels of p14ARF and p16INK4A. Together our data indicate that BMI1 expression is required for maintenance and self-renewal of normal and leukemic stem and progenitor cells, and that expression of BMI1 protects cells against oxidative stress.

scholarly journals STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells

Blood ◽  
2007 ◽  
Vol 110 (8) ◽  
pp. 2880-2888 ◽  
Author(s):  
Hein Schepers ◽  
Djoke van Gosliga ◽  
Albertus T. J. Wierenga ◽  
Bart J. L. Eggen ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Fold Reduction ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Precise Role ◽  
Term Maintenance

Abstract The transcription factor STAT5 fulfills a distinct role in the hematopoietic system, but its precise role in primitive human hematopoietic cells remains to be elucidated. Therefore, we performed STAT5 RNAi in sorted cord blood (CB) and acute myeloid leukemia (AML) CD34+ cells by lentiviral transduction and investigated effects of STAT5 downmodulation on the normal stem/progenitor cell compartment and the leukemic counterpart. STAT5 RNAi cells displayed growth impairment, without affecting their differentiation in CB and AML cultures on MS5 stroma. In CB, limiting-dilution assays demonstrated a 3.9-fold reduction in progenitor numbers. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays, and the average LTC-IC frequency was 3.25-fold reduced from 0.13% to 0.04% by STAT5 down-regulation. Single-cell sorting experiments of CB CD34+/CD38− cells demonstrated a 2-fold reduced cytokine-driven expansion, with a subsequent 2.3-fold reduction of progenitors. In sorted CD34+ AML cells with constitutive STAT5 phosphorylation (5/8), STAT5 RNAi demonstrated a reduction in cell number (72% ± 17%) and a decreased expansion (17 ± 15 vs 80 ± 58 in control cultures) at week 6 on MS5 stroma. Together, our data indicate that STAT5 expression is required for the maintenance and expansion of primitive hematopoietic stem and progenitor cells, both in normal as well as leukemic hematopoiesis.

The Neuropeptides Orexin a and B Have An Impact on Functional Properties of Human CD34+ Stem and Progenitor Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1393-1393
Author(s):  
Ingmar Bruns ◽  
Patrick Cadeddu ◽  
Sebastian Büst ◽  
Boris Goerg ◽  
Johannes C Fischer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Orexin A ◽  
Long Term Culture ◽  
Human Cd34 ◽  
Orexin Receptors ◽  
Cd34 Cells ◽  
Proliferation And Apoptosis ◽  
Stem And Progenitor Cells

Abstract Orexin receptors play a role in regulation of sleep-wake-rhythm, food intake and energy homeostasis and they were long thought to be exclusively expressed in the nervous system. During the last years orexin receptors are being identified in a growing number of peripheral tissues. We have earlier detected orexin receptor 1 and 2 expression on human CD34+ blood stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seem to be restricted to the central nerve system to this date. The main downstream signaling pathways of the orexin receptors include Ca2+-dependent signaling associated with activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways. In an attempt to investigate if the receptors are functionally active in CD34+ stem and progenitor cells, we used live cell calcium imaging and stimulated purified CD34+ stem and progenitor cells with orexin A and B. Upon stimulation a massive intracellular calcium release was seen which could not been detected using cells preincubated with the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the selective OX1R-Antagonist SB334867 and CD34 negative cells. Additionally, upon stimulation with orexin A and B we found ERK (1/2) activation at a maximum 3 hours following incubation with orexin A whereas no effect was seen after stimulation with orexin B. To investigate a potential impact on the functional properties of human CD34+ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. So far, no effects of orexin stimulation on the proliferation and apoptosis of CD34+ cells were apparent. Remarkably, stimulation with orexin A and B led to a significantly higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors BFU-E (burst forming unit erythrocyte) and CFU-E (colony forming unit erythrocyte). A more immature phenotype of orexin-stimulated CD34+ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significantly higher frequency of LTC-IC (long-term-culture initiating cells) indicating also a more immature phenotype of orexin-stimulated cells and a greater repopulating capacity. The selective orexin receptor antagonist SB-334867 abrogated these effects. No differences could be observed regarding the migration towards SDF-1 with and without stimulation with orexin A and B. Still, orexin A and B led to a decrease in the adhesive capacity of CD34+ stem and progenitor cells to fibronectin coated dishes. Since orexin receptors are coupled to inhibitory G-proteins (Gi/q) and stimulatory G-proteins (Gs) dependent on the tissue, we incubated CD34+ cells with the selective inhibitor of Gi – proteins pertussis toxin concurrently to stimulation with orexins and observed no differences in the adhesive capacity of CD34+ cells compared to the unstimulated controls suggesting coupling of the orexin receptor 1 and 2 to Gi – proteins rather than Gs-proteins in CD34+ cells. Given this functional impact of the orexin system on CD34+ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34+ cells or if they are humorally transported to the bone marrow cavity. Using ELISA we did not find autocrine production of orexin by CD34+ cells whereas orexin could be detected in the serum obtained by bone marrow biopsies and peripheral blood pointing rather towards a humoral delivery of orexins to CD34+ cells. Taken together, our findings indicate a functional role of the orexin system in CD34+ stem and progenitor cells.

Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1329-1329
Author(s):  
Aleksandra Rizo ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
Jan Jacob Schuringa
Keyword(s):  
Cord Blood ◽  
Cell Expansion ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC). In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors. After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.

Long-Term Expansion and Self Renewal Is Contained in the CD34+, but Not the CD34- Subfraction of Nucleophosmin Mutated Acute Myeloid Leukemia Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1349-1349
Author(s):  
Carolien Woolthuis ◽  
Lina Han ◽  
Djoke van Gosliga ◽  
Philip Kluin ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hox Genes ◽  
Mutant Protein ◽  
Self Renewal ◽  
Cd34 Cells ◽  
Stromal Layer ◽  
Acute Myeloid

Abstract Mutations in the nucleophosmin (NPM) gene are found in about 30% of cases of acute myeloid leukemia (AML) and lead to a dislocation of the nucleophosmin protein from the nucleus to the cytoplasm (NPMc+ AML). NPMc+ AML shows distinctive biological and clinical features, including a unique gene expression profile, a distinct microRNA signature, low percentage of CD34+ cells, increased incidence of Flt3-ITD (about 40% of cases), good response to induction chemotherapy and (in the absence of Flt3-ITD) a favourable prognosis. Despite significant progress in the characterization of the NPMc+ AML subgroup, questions remain about the leukemia-initiating cell. Distinct features of NPMc+ AML, including multilineage involvement and overexpression of HOX-genes, may point to an early progenitor as the leukemia-initiating cell, but the characteristic low percentage of CD34+ cells may point to a more differentiated leukemic stem cell in NPMc+ AML. To gain more insight in the leukemia-initiating cell in AML with mutated NPM, NPMc+ AML cells were sorted based on the expression of CD34 (n=8, the percentage of CD34+ in the total AML fraction varied between 0.06 and 37%). Western blotting, using an antibody that specifically recognizes the nucleophosmin mutant protein revealed that the NPM mutant protein is expressed in both CD34+ and CD34− cells, proving that the CD34+ NPMc+ AML cells belong to the leukemic clone. This was verified by sequencing the NPM gene in CD34+ and CD34− AML cells. Importantly, culture of sorted CD34+ and CD34− NPMc+ AML cells on a stromal layer revealed that the CD34+ but not the CD34− cells of NPMc+ AML were capable of expanding and initiating long-term growth. In the first 5 weeks of culture an at least 16 fold (range 16–208) expansion of CD34+ AML cells was seen in 5 out of 6 NPMc+ AML cases. This expansion was associated with the formation of cobblestone areas (CAs) under the stromal layer within 3 weeks after plating. The NPMc+ AML cells which expanded in culture were able to expand further after replating in 4 out of 5 investigated cases (fold expansion range 1.6–2.5), indicative of the self renewal capacity of these CD34+ NPMc+ AML cells. Gene expression analysis of CD34+ and CD34− NPMc+ AML cells of 4 cases analyzed thus far revealed the presence of the characteristic HOX-overexpression profile in both CD34+ and CD34− NPMc+ AML cells. In summary, this study shows that the NPM mutation is not only present in CD34−, but also in CD34+ cells of NPMc+ AML and that the properties of long-term expansion and self renewal belong exclusively to the CD34+ subfraction of NPMc+ AML.

Malignant Myeloma Cells Impair Phenotype and Function of stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1799-1799
Author(s):  
Ingmar Bruns ◽  
Sebastian Büst ◽  
Akos G. Czibere ◽  
Ron-Patrick Cadeddu ◽  
Ines Brückmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Plasma Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Stem And Progenitor Cells

Abstract Abstract 1799 Poster Board I-825 Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established. In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E). Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors. Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level. Disclosures Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

The Tetraspanin CD82 Defines Erythroid Committment, Regulates Stem- and Progenitor Cell Survival and Mediates Adhesion.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4156-4156
Author(s):  
Andreas Burchert ◽  
Barbara Denecke ◽  
Tobias Haerle ◽  
Andreas Neubauer
Keyword(s):  
Progenitor Cells ◽  
T Cell Activation ◽  
Cell Biology ◽  
Cell Activation ◽  
Isotype Control ◽  
Cd34 Cells ◽  
Colony Forming Units ◽  
Stem And Progenitor Cells

Abstract CD82 belongs to the tetraspanin superfamily, members of which regulate multiple aspects of cell biology such as T-cell activation, proliferation, differentiation, and adhesion.We have previously shown expression of CD82 on hematopoietic progenitor cells. Here we were seeking to shed light on the functional role of CD82 in hematopoiesis. First, we found that CD82 expression is strongly associated with an erythroid committment as exclusively the CD82bright fraction but not the CD82medium or CD82dim fraction of CD34+ cells gave rise to erythroid colony forming units (CFU). In order to manipulate CD82 activity, we used as surrogate ligand, a previously described, CD82-activating monoclonal antibody (moAb), clone 50F11, to activate CD82 on hematopoietic precursors. We found that 50F11, but not another CD82 specific antibody clone, BL-2, specifically induced tyrosine phosphorylation of CD34+ cells. In Dexter-type long-term cultures (D-LTC) 50F11, and not IgG1 isotype control moAbs significantly inhibited myelopoiesis and the number of CD34+ clonogenic progenitors. Moreover, in long term culture initiating cell (LTC-IC) assays, 50F11as compared to isotype control antibodies substantially inhibited, but not entirely abolished, the number of 5 week-LTC-IC′s, indicating that CD82 activation inhibits progenitor - and stem cell proliferation or self renewal. Finally, plastic immobilized 50F11-antibodies caused a time-, and concentration dependent induction of adhesion of CD34+ cells, which was associated with the formation of F-actin and development of multipolar extensions. Finally, CD82 ligation by 50F11 caused a statistically significant down-regulation of the integrin CD49d (p=0,036) and CD62L (p=0,010). Together, it is shown that CD34+/CD82high cells characterize an erythroid committment implying a role for this tetraspanin in erythroid hematopoiesis. Activation of CD82 induces adhesion and negatively regulates proliferation of adult stem- and progenitor cells. This implicates a so far unknown role for CD82 in the regulation of early hematopoiesis.

STAT5-Induced Self-Renewal and Impaired Myelopoiesis of Human Hematopoietic Stem/Progenitor Cells Involves Downmodulation of C/EBPα.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 268-268
Author(s):  
Jan Jacob Schuringa ◽  
Bart-Jan Wierenga ◽  
Hein Schepers ◽  
Malcolm A.S. Moore ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Rt Pcr ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Affymetrix Microarray ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Cd34 Cells ◽  
Proliferative Advantage

Abstract Previously, we demonstrated that enforced activation of STAT5 in human cord blood (CB)-derived stem/progenitor cells results in enhanced long-term stem cell self-renewal and impaired myelopoiesis (J.J.Schuringa et al, J.Exp.Med. 2004;200:623). Now, C/EBPα was identified as a critical transcription factor that is downregulated by STAT5. Affymetrix microarray analysis on STAT5A(1*6)-transduced CD34+ cells identified C/EBPα as the most prominently downregulated gene (−3.3 fold), and these data were confirmed by RT-PCR and Western blotting. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPα-ER protein was co-expressed with the STAT5A(1*6) mutant in CB CD34+ cells by using a retroviral approach. Re-expression of C/EBPα in STAT5A(1*6) cells resulted in a marked restoration of myelopoiesis as determined by morphological analyses, FACS analyses for myeloid markers such as CD11b, CD14 and CD15, and RT-PCR for myeloid-restricted genes such as g-csfr. While enforced activation of STAT5A resulted in accelerated erythropoiesis, this was blocked when C/EBPα was re-introduced into STAT5A(1*6) cells. Similarly, the proliferative advantage imposed on CD34+ cells by STAT5A(1*6) depended on the downmodulation of C/EBP as reintroduction of C/EBPα in these cells induced a quick cell cycle arrest and the onset of myeloid differentiation. At the stem/progenitor cell level, LTC-IC frequencies were elevated from 0.5% to 11% by STAT5A(1*6) as compared to controls, but these elevated LTC-IC frequencies were strongly reduced when C/EBPα was reintroduced in STAT5A(1*6) cells. Enumeration of progenitors in methylcellulose assays revealed similar results, the number of CFCs was reduced over 10-fold when C/EBPα was expressed in STAT5A(1*6) cells. Also, secondary CAFCs and long-term cultures could only be generated from STAT5A(1*6) expressing cells, but not from cells that co-expressed STAT5A(1*6) and C/EBPα. Taken together, these data indicate that STAT5-induced self-renewal and impaired myelopoiesis involves downmodulation of C/EBPα.

Optimization of the Sleeping Beauty Transposon System to Achieve Stable Transgene Expression in Human CD34+ Hematopoietic Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3527-3527
Author(s):  
Teiko Sumiyoshi ◽  
Roger P Hollis ◽  
Nathalia Holt ◽  
Donald B. Kohn
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Sleeping Beauty ◽  
Egfp Expression ◽  
Stable Gene ◽  
Human Cd34 ◽  
Gfp Reporter ◽  
Cd34 Cells

Abstract Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. Transposon-mediated gene integration may have advantages over viral vectors, with a greater transgene carrying capacity and potentially safer integration site profile. Due to these characteristics of SB, there has been great interest in its potential use in hematopoietic stem cell (HSC) gene therapy. In this study, we optimized the SB transposon-mediated gene transfer system to achieve higher stable transgene expression in K562 human erythroleukemia cells, Jurkat human T-lymphoid cells, and primary human CD34+ hematopoietic progenitor cells. The SB transposon system was optimized by two approaches: to increase the transposition efficacy, a hyperactive mutant of SB, HSB16, was used (Baus et al.; Mol Ther12:1148, 2005); to optimize the expression of the SB transposase and the transgene cassette carried by the transposon, three different viral and cellular promoters were evaluated, including the modified MPSV long terminal repeat (MNDU3) enhancer-promoter, the human cytomegalovirus (hCMV) immediate-early region enhancer-promoter, and the human elongation factor 1 (hEF1a) promoter. SB components were delivered in trans into the target cells by nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression using fluorescent-activated cell sorting (FACS) analysis over 3–4 weeks. The functional assay showed that HSB16 was a more efficient enzyme compared to the original SB. In purified human cord blood CD34+ cells, HSB16 achieved nearly 7-fold higher long-term transgene expression with 90% less plasmid DNA (from 10 mcg of SB reduced to 1 mcg of HSB16) than the original SB transposase. The highest level of stable transgene integration in all three cell types was achieved using the hEF1a promoter to express HSB16 in comparison to either the hCMV or MND promoter. Our data also suggested that optimal GFP reporter gene expression from the integrated transposon was influenced by the type of promoter and the target cell type. Significantly higher levels of eGFP expression (5-fold) were achieved with the hEF1a promoter in Jurkat human T cells, compared to that achieved with the MND promoter; in contrast the MND promoter expressed GFP at the highest level in K562 myeloid cells. In primary human CD34+ cord blood progenitors, optimal transgene integration and expression was achieved using the hEF1a promoter to express the SB transposase combined with the MND promoter to express GFP reporter, when studied under conditions directing myeloid differentiation. Stable transgene expression was achieved at levels up to 27% for over 4 weeks after optimized gene transfer to CD34+ cells (ave=17%, n=4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34+ progenitor cells are currently in progress. In conclusion, the optimized SB transposon system in primary human CD34+ hematopoietic progenitors reported here has improved the stable gene transfer efficiency by 29-fold, compared to our prior published data (< 1% - Hollis et al.; Exp Hematol34:1333, 2006). The long-term stable gene expression achieved by our optimized SB transposon system shows promise for further advancement of non-viral based HSC gene therapy.

Overexpression of Oncogenic KRAS G12V in Human Stem and Progenitor Cells Enhances Proliferation and Initiates Monocytic Differentiation Via Intrinsic and Extrinsic Pathways.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3975-3975
Author(s):  
Szabolcs Fatrai ◽  
Djoke van Gosliga ◽  
Lina Han ◽  
Simon M. G. J. Daenen ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Signal Transduction Pathways ◽  
Monocytic Differentiation ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells ◽  
P38 Pathway

Abstract Abstract 3975 Poster Board III-911 In human hematopoietic malignancies, Ras mutations are frequently present in monocytic and T-cell leukemias. In this study we investigated KRAS G12V-induced phenotypes in human stem and progenitor cells and identified signal transduction pathways that are involved. Using a retroviral expression system, KRAS G12V was introduced to human CD34+ cord blood (CB) cells and proliferation, differentiation and stem cell/progenitor frequencies were evaluated. Overexpression of constitutively active KRAS G12V induced a strong increase in cell expansion over 5-fold in MS5 bone marrow stromal cocultures as well as in cytokine-driven liquid cultures, which coincided with increased early cobblestone formation and induction of monocytic differentiation. Erythroid progenitors were greatly reduced by introduction of KRAS G12V and Q-PCR analysis revealed that expression of PU.1 was increased in conjunction with reduced GATA1 expression in KRAS G12V cells. Progenitor frequencies were increased 6-fold in KRAS-transduced cells within 1 week after plating on MS5. By week three progenitors were exhausted and KRAS-transduced cells were terminally differentiated into monocytes/macrophages. These results were in line with the strong reduction in LTC-IC frequencies at week 5, indicating that also the stem cell pool was exhausted. Intriguingly, when KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, we observed that the non-transduced cells also displayed a strong growth advantage, coinciding with enhanced early cobblestone formation. Furthermore, the addition of conditioned medium from KRAS G12V-transduced cells grown on MS5 to non-transduced CB cells induced a strong growth advantage and formation of early CAFCs. These observations indicate that, besides intrinsic pathways, secreted factor(s) play an important role in the phenotypes induced by KRAS G12V in human CB CD34+ cells. Current studies include mass-spectroscopy analysis of the secretome of KRAS G12V-transduced CB CD34+ cells to identify the factor(s) that are involved. In order to elucidate signal transduction pathways that mediate KRAS G12V-induced phenotypes, Western-blot analysis was performed. These experiments revealed an increase in phospho-ERK1/2, phospho-p38 and phospho- STAT5 (Y694) levels in KRAS-transduced cells, whereas phospho-JNK was not induced and phospho-C/EBPa (S21) levels were slightly reduced. Induction of STAT5 Y649 phosphorylation by KRAS G12V was confirmed by intracellular phosphoFACS analysis, whereby both in HSCs as well as in more committed MPPs KRAS-induced phosphorylation of STAT5 was observed. KRAS-transduced cells did not show GM-CSF hypersensitivity in any measured cell population upon activation. Inhibition of the ERK/MAPK pathway using the MEK inhibitor U0126 resulted in strongly reduced expansion in MS5 cocultures, whereby both intrinsically induced proliferation as well as proliferation induced via secreted factor(s) were impaired. KRAS G12V-induced monocytic differentiation was not significantly affected by MEK inhibition. While inhibition of the JNK pathway hardly affected proliferation and differentiation of KRAS G12V cells, inhibition of the p38 pathway using SB203580 inhibitor impaired both proliferation and differentiation. When KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, inhibition of p38 predominantly affected the transduced cells but not the non-transduced cells, suggesting that the p38 pathway particularly mediates intrinsic phenotypes imposed by KRAS G12V. In conclusion, we show that overexpression of oncogenic KRAS G12V in human CD34+ cells enhances proliferation and initiates monocytic differentiation via intrinsic and extrinsic pathways. Disclosures: No relevant conflicts of interest to declare.

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