In vivo expansion of gene-modified hematopoietic cells by a novel selective amplifier gene utilizing the erythropoietin receptor as a molecular switch

2004 ◽  
Vol 6 (1) ◽  
pp. 22-31 ◽  
Author(s):  
Takeyuki Nagashima ◽  
Yasuji Ueda ◽  
Yutaka Hanazono ◽  
Akihiro Kume ◽  
Hiroaki Shibata ◽  
...  
Keyword(s):  
Hematopoietic Cells ◽  
Molecular Switch ◽  
Erythropoietin Receptor ◽  
Selective Amplifier

scholarly journals Development of a Novel Selective Amplifier Gene for Controllable Expansion of Transduced Hematopoietic Cells

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3884-3892 ◽  
Author(s):  
Keiko Ito ◽  
Yasuji Ueda ◽  
Masaki Kokubun ◽  
Masashi Urabe ◽  
Toshiya Inaba ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Selective Amplifier ◽  
Marrow Cells

Abstract To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)–dependent murine cell line, Ba/F3, the cells showed IL-3–independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Δ(5-195)GCRER, whose product lacks the extracellular G-CSF–binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Δ(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

In vivo expansion of transduced murine hematopoietic cells with a selective amplifier gene

2003 ◽  
Vol 5 (3) ◽  
pp. 175-181 ◽  
Author(s):  
Akihiro Kume ◽  
Masahide Koremoto ◽  
Ruifang Xu ◽  
Takashi Okada ◽  
Hiroaki Mizukami ◽  
...  
Keyword(s):  
Hematopoietic Cells ◽  
Selective Amplifier

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

Neuropilin-1 on hematopoietic cells as a source of vascular development

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1801-1809 ◽  
Author(s):  
Yoshihiro Yamada ◽  
Yuichi Oike ◽  
Hisao Ogawa ◽  
Yasuhiro Ito ◽  
Hajime Fujisawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fetal Liver ◽  
Vascular Development ◽  
Hematopoietic Cells ◽  
Vegf Receptor ◽  
Knock Out ◽  
Neuropilin 1 ◽  
Vasculogenesis And Angiogenesis

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-165 (VEGF165) and acts as a coreceptor that enhances the function of VEGF165 through VEGF receptor-2 (VEGFR-2). Studies using transgenic and knock-out mice of NP-1 indicated that this molecule is important for vascular development as well as neuronal development. We recently reported that clustered soluble NP-1 phosphorylates VEGFR-2 on endothelial cells with a low dose of VEGF165 and rescues the defective vascularity of the NP-1−/− embryo in vitro and in vivo. Here we show that NP-1 is expressed by CD45+ hematopoietic cells in the fetal liver, can bind VEGF165, and phosphorylates VEGFR-2 on endothelial cells. CD45+NP-1+ cells rescued the defective vasculogenesis and angiogenesis in the NP-1−/− P-Sp (para-aortic splanchnopleural mesodermal region) culture, although CD45+NP-1− cells did not. Moreover, CD45+NP-1+ cells together with VEGF165 induced angiogenesis in an in vivo Matrigel assay and cornea neovascularization assay. The extracellular domain of NP-1 consists of “a,” “b,” and “c” domains, and it is known that the “a” and “c” domains are necessary for dimerization of NP-1. We found that both the “a” and “c” domains are essential for such rescue of defective vascularities in the NP-1 mutant. These results suggest that NP-1 enhances vasculogenesis and angiogenesis exogenously and that dimerization of NP-1 is important for enhancing vascular development. In NP-1−/− embryos, vascular sprouting is impaired at the central nervous system (CNS) and pericardium where VEGF is not abundant, indicating that NP-1–expressing cells are required for normal vascular development.

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

scholarly journals WASP confers selective advantage for specific hematopoietic cell populations and serves a unique role in marginal zone B-cell homeostasis and function

Blood ◽  
2008 ◽  
Vol 112 (10) ◽  
pp. 4139-4147 ◽  
Author(s):  
Lisa S. Westerberg ◽  
Miguel A. de la Fuente ◽  
Fredrik Wermeling ◽  
Hans D. Ochs ◽  
Mikael C. I. Karlsson ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Hematopoietic Cells ◽  
Selective Advantage ◽  
Relative Contribution ◽  
B Cell Homeostasis ◽  
And Function

Abstract Development of hematopoietic cells depends on a dynamic actin cytoskeleton. Here we demonstrate that expression of the cytoskeletal regulator WASP, mutated in the Wiskott-Aldrich syndrome, provides selective advantage for the development of naturally occurring regulatory T cells, natural killer T cells, CD4+ and CD8+ T lymphocytes, marginal zone (MZ) B cells, MZ macrophages, and platelets. To define the relative contribution of MZ B cells and MZ macrophages for MZ development, we generated wild-type and WASP-deficient bone marrow chimeric mice, with full restoration of the MZ. However, even in the presence of MZ macrophages, only 10% of MZ B cells were of WASP-deficient origin. We show that WASP-deficient MZ B cells hyperproliferate in vivo and fail to respond to sphingosine-1-phosphate, a crucial chemoattractant for MZ B-cell positioning. Abnormalities of the MZ compartment in WASP−/− mice lead to aberrant uptake of Staphylococcus aureus and to a reduced immune response to TNP-Ficoll. Moreover, WASP-deficient mice have increased levels of “natural” IgM antibodies. Our findings reveal that WASP regulates both development and function of hematopoietic cells. We demonstrate that WASP deficiency leads to an aberrant MZ that may affect responses to blood-borne pathogens and peripheral B-cell tolerance.

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