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Objective: Adenoviral vector-mediated gene expression is an attractive approach to manipulate or report gene expression in human hematopoietic stem cells (HSC) when transient gene expression is preferred. Previous studies have demonstrated that fiber-retargeted Ad5F35 vectors can mediate efficient gene transfer into human HSCs. In this study, we investigated the potential of bi-directional promoter-controlled Ad5F35 vector for coordinated dual gene expression in candidate HSCs., Materials and Methods: We have engineered Ad5F35-DeltaLNGFR-BiDp encoding kinase domain deleted low-affinity NGF receptor (DeltaLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter, which is composed of human phosphoglycerate kinase promoter and minimal core promoter from human cytomegalovirus. The expression pattern of DeltaLNGFR and GFP following Ad5F35-DeltaLNGFR-BiDp gene transfer in various cell types, including candidate HSCs, was compared to Ad5F35-DeltaLNGFR-IRES vector encoding phosphoglycerate kinase promoter-controlled bicistronic expression cassette for DeltaLNGFR and GFP., Results: Using Ad5F35-DeltaLNGFR-BiDp, we demonstrated a coordinated, high-level dual gene expression in leukemic cells and cord blood CD34(+) cells. However, the ability of Ad5F35-DeltaLNGFR-BiDp-GFP for coordinated dual gene expression varied significantly between repopulating progenitor cells. In nonobese diabetic severe combined immune deficient mice bone marrow transplantation assay, sorted CD34(+)/DeltaLNGFR(+)/GFP(+) cells following infection with Ad5F35-DeltaLNGFR-BiDp showed predominantly myeloid lineage reconstitution with limited lymphoid lineage differentiation capacity, whereas the CD34(+)/DeltaLNGFR(+)/GFP(-) cells exhibited both myeloid and lymphoid reconstitution., Conclusions: This study indicates that bi-directional promoter-controlled Ad5F35 vector, such as Ad5F35-DeltaLNGFR-BiDp, can be particularly useful for manipulation of myeloid progenitor cells and potentially in myeloid lineage leukemic cells as well., (Copyright 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Current approaches for treatment of late-stage breast cancer rarely result in a long-term cure. In part this is due to tumor stroma that prevents access of systemically or intratumorally applied therapeutics. We propose a stem cell gene therapy approach for controlled tumor stroma degradation that uses the pathophysiologic process of recruitment of inflammatory cells into the tumor. This approach involves genetic modification of hematopoietic stem cells (HSCs) and their subsequent transplantation into tumor-bearing mice. We show that inducible, intratumoral expression of relaxin (Rlx) either by transplanting tumor cells that contained the Rlx gene or by transplantation of mouse HSCs transduced with an Rlx-expressing lentivirus vector delays tumor growth in a mouse model of breast cancer. The antitumor effect of Rlx was mediated through degradation of tumor stroma, which provided increased access of infiltrating antitumor immune cells to their target tumor cells. Furthermore, we have shown in a human/mouse chimeric model that genetically modified HSCs expressing a transgene can access the tumor site. Our findings are relevant for cancer gene therapy and immunotherapy.

Objective: The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells., Materials and Methods: We investigated and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood., Results: Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under in vitro conditions favoring B-cell differentiation, neither P190 nor P210 BCR/ABL1-expressing cells formed detectable levels of CD19-positive cells. Gene expression profiling revealed that P190 BCR/ABL1 and P210 BCR/ABL1 induced almost identical gene expression profiles., Conclusions: Our data suggest that the early cellular and transcriptional effects of P190 BCR/ABL1 and P210 BCR/ABL1 expression are very similar when they are expressed in the same human progenitor cell population, and that STAT5 is an important regulator of BCR/ABL1-induced erythroid cell expansion.

The proliferation and differentiation of primitive hematopoietic cells is tightly controlled by a number of signaling pathways. Transient blockage or enhancement of these signaling pathways may provide a new approach to manipulate the proliferation and differentiation of primitive hematopoietic cells. Adenoviral vectors have in recent years emerged as powerful tools for transient gene expression in human primitive hematopoietic cells. Important advantageous properties of adenoviral vectors include: feasible production of high-titer vector preparations, high efficiency in transducing both quiescent and actively dividing cells, high levels of transient gene expression, and a lack of mutagenic properties associated with integrating vectors. Progress in adenoviral fiber retargeting was recently demonstrated to enable high gene transfer efficiency into nondividing human CD34(+) cells and nonobese diabetic/severe combined immunodeficient mouse bone marrow repopulating cells (SRCs), via the ubiquitously expressed CD46 as a cellular receptor. Importantly, fiber-retargeted adenoviral vectors can be engineered to report gene expression in single living CD34(+) cells, thereby facilitating the isolation and characterization of SRCs and its downstream progenitors based on intrinsic signaling pathways. This review focuses on the current progress and the potential future applications of adenoviral gene transfer into human primitive hematopoietic cells and leukemic cells.

Telomerase activity has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. However, it has been unclear whether human HSCs have telomerase activity and how telomerase activity is regulated within the HSC and progenitor pool. Here, we isolated living cord-blood (CB) CD34(+) cells with up-regulated human telomerase reverse transcriptase (hTERT) expression by using an hTERT-reporting adenoviral vector encoding destabilized green fluorescent protein (dGFP) driven by the hTERT promoter, and functionally characterized them in comparison with control vector-transduced CD34(+) cells expressing GFP. Following a 2-day serum-free transduction protocol, cells were sorted into a dGFP(+) and a GFP(+) fraction. Cell-cycle analysis revealed that the dGFP(+) cells had a greater proportion of cells in S/G(2)/M phase compared with the GFP(+) cells, (56% +/- 1.8% vs 35% +/- 4.3%; P < .001) and fewer cells in G(0) phase (8.1% +/- 3.0% vs 20% +/- 4.7%; P < .01) However, the colony-forming and short-term nonobese diabetic/severe combined immunodeficient (NOD/SCID) B2m(-/-) mice bone marrow-repopulating capacities were similar between the dGFP(+) and the GFP(+) cells. Interestingly, the dGFP(+) cells had a 6-fold lower repopulating capacity in NOD/SCID mice compared with the GFP(+) cells and lacked secondary NOD/SCID B2m(-/-) mice bone marrow-repopulating capacity. Thus, up-regulation of hTERT expression within the CB HSC pool is accompanied by decreased self-renewal capacity.

Studies in vitro implicate transforming growth factor beta (TGF-beta) as a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. In vivo studies have been hampered by early lethality of knock-out mice for TGF-beta isoforms and the receptors. To directly assess the role of TGF-beta signaling for hematopoiesis and hematopoietic stem cell (HSC) function in vivo, we generated a conditional knock-out model in which a disruption of the TGF-beta type I receptor (T beta RI) gene was induced in adult mice. HSCs from induced mice showed increased proliferation recruitment when cultured as single cells under low stimulatory conditions in vitro, consistent with an inhibitory role of TGF-beta in HSC proliferation. However, induced T beta RI null mice show normal in vivo hematopoiesis with normal numbers and differentiation ability of hematopoietic progenitor cells. Furthermore HSCs from T beta RI null mice exhibit a normal cell cycle distribution and do not differ in their ability long term to repopulate primary and secondary recipient mice following bone marrow transplantation. These findings challenge the classical view that TGF-beta is an essential negative regulator of hematopoietic stem cells under physiologic conditions in vivo.

Retroviral overexpression of the transcription factor HOXB4 results in a rapid increase in proliferation of murine hematopoietic stem cells both in vivo and in vitro. Therefore, we asked whether transient overexpression of HOXB4 would increase proliferation of human primitive hematopoietic progenitors. Transient overexpression of HOXB4 was generated in umbilical cord blood (CB) CD34(+) cells by a recombinant adenovirus (AdHOXB4) expressing HOXB4 together with the enhanced green fluorescent protein (GFP). Transduced, GFP(+) cells were cultured in serum-free medium containing cytokines that primarily support the growth of primitive hematopoietic progenitors. In contrast to previous findings using retroviral overexpression of HOXB4, we did not observe any increase in proliferation of primitive progenitors or increased colony formation of clonogenic progenitors, including progenitor progeny from long-term culture-initiating cells following adenoviral vector overexpression of HOXB4 in CB CD34(+) cells. However, enforced expression of HOXB4 by the adenoviral vector significantly increased myeloid differentiation of primitive hematopoietic progenitors. Since retroviral vectors generate low and continuous levels of transgene expression in contrast to the high, transient levels generated by the adenoviral vector, our findings suggest that the high levels of HOXB4 expression generated by AdHOXB4 in human CB CD34(+) cells direct the cells toward a myeloid differentiation program rather than increased proliferation.

Hemopoietic stem cells (HSCs) are maintained at relative quiescence by the balance between the positive and negative regulatory factors that stimulate or inhibit their proliferation. Blocking the action of negative regulatory factors may provide a new approach for inducing HSCs into proliferation. A variety of studies have suggested that TGF-beta negatively regulates cell cycle progression of HSCs. In this study, a dominant negatively acting mutant of TGF-beta type II receptor (TbetaRIIDN) was transiently expressed in HSCs by using adenoviral vector-mediated gene delivery, such that the effects of disrupting the autocrine TGF-beta signaling in HSCs can be directly examined at a single cell level. Adenoviral vectors allowing the expression of TbetaRIIDN and green fluorescence protein in the same CD34(+)CD38(-)Lin(-) cells were constructed. Overexpression of TbetaRIIDN specifically disrupted TGF-beta-mediated signaling. Autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells was studied in single cell assays under serum-free conditions. Transient blockage of autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells enhanced their survival. Furthermore, the overall proliferation potential and proliferation kinetics in these cells were significantly enhanced compared with the CD34(+)CD38(-)Lin(-) cells expressing green fluorescence protein alone. Therefore, we have successfully blocked the autocrine TGF-beta-negative regulatory loop of primitive hemopoietic progenitor cells.

Retroviral overexpression of the transcription factor HOXB4 results in a rapid increase in proliferation of murine hematopoietic stem cells both in vivo and in vitro. Therefore, we asked whether transient overexpression of HOXB4 would increase proliferation of human primitive hematopoietic progenitors. Transient overexpression of HOXB4 was generated in umbilical cord blood (CB) CD34+ cells by a recombinant adenovirus (AdHOXB4) expressing HOXB4 together with the enhanced green fluorescent protein (GFP). Transduced, GFP+ cells were cultured in serum-free medium containing cytokines that primarily support the growth of primitive hematopoietic progenitors. In contrast to previous findings using retroviral overexpression of HOXB4, we did not observe any increase in proliferation of primitive progenitors or increased colony formation of clonogenic progenitors, including progenitor progeny from long-term culture-initiating cells following adenoviral vector overexpression of HOXB4 in CB CD34+ cells. However, enforced expression of HOXB4 by the adenoviral vector significantly increased myeloid differentiation of primitive hematopoietic progenitors. Since retroviral vectors generate low and continuous levels of transgene expression in contrast to the high, transient levels generated by the adenoviral vector, our findings suggest that the high levels of HOXB4 expression generated by AdHOXB4 in human CB CD34+ cells direct the cells toward a myeloid differentiation program rather than increased proliferation. [Copyright &y& Elsevier]