Journal articles on the topic 'Immunodeficienza primitiva'
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1
Dekate, Jyoti, and Runjan Chetty. "Epstein-Barr Virus–Associated Smooth Muscle Tumor." Archives of Pathology & Laboratory Medicine 140, no. 7 (July 1, 2016): 718–22. http://dx.doi.org/10.5858/arpa.2015-0120-rs.
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Immunodeficient individuals are prone to develop a number of opportunistic infections and unique neoplasms. Epstein-Barr virus–associated smooth muscle tumor is an uncommon neoplasm associated with immunodeficiency. It has been described in patients infected with human immunodeficiency virus, in the posttransplant setting, and in those with congenital immunodeficiency. Different anatomic sites can be involved by Epstein-Barr virus–associated smooth muscle tumor, and even multiple locations can contain these unique lesions within the same patient. The presence of variable numbers of intratumoral lymphocytes and primitive round cell areas are the unique defining features for this tumor. Histopathologic features may vary considerably in terms of cellular atypia, mitotic activity, and necrosis, with no correlation to the clinical behavior. Demonstration of Epstein-Barr virus infection by in situ hybridization within tumor cell remains critical for the diagnosis. The mechanism for Epstein-Barr virus infection of progenitor cells and neoplastic transformation has been an area of interest and conjecture. Different treatment strategies are proposed according to underlying disease status. This paper reviews the clinicopathologic features of this uncommon neoplasm with detailed discussion of the role of Epstein-Barr virus in the pathogenesis.
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van der Loo, Johannes C. M., Helmut Hanenberg, Ryan J. Cooper, F. Y. Luo, Emmanuel N. Lazaridis, and David A. Williams. "Nonobese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) Mouse as a Model System to Study the Engraftment and Mobilization of Human Peripheral Blood Stem Cells." Blood 92, no. 7 (October 1, 1998): 2556–70. http://dx.doi.org/10.1182/blood.v92.7.2556.
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Abstract Mobilized CD34+ cells from human peripheral blood (PB) are increasingly used for hematopoietic stem-cell transplantation. However, the mechanisms involved in the mobilization of human hematopoietic stem and progenitor cells are largely unknown. To study the mobilization of human progenitor cells in an experimental animal model in response to different treatment regimens, we injected intravenously a total of 92 immunodeficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with various numbers of granulocyte colony-stimulating factor (G-CSF) –mobilized CD34+ PB cells (ranging from 2 to 50 × 106cells per animal). Engraftment of human cells was detectable for up to 6.5 months after transplantation and, depending on the number of cells injected, reached as high as 96% in the bone marrow (BM), displaying an organ-specific maturation pattern of T- and B-lymphoid and myeloid cells. Among the different mobilization regimens tested, human clonogenic cells could be mobilized from the BM into the PB (P= .019) with a high or low dose of human G-CSF, alone or in combination with human stem-cell factor (SCF), with an average increase of 4.6-fold over control. Therefore, xenotransplantation of human cells in NOD/SCID mice will provide a basis to further study the mechanisms of mobilization and the biology of the mobilized primitive human hematopoietic cell.
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van der Loo, Johannes C. M., Helmut Hanenberg, Ryan J. Cooper, F. Y. Luo, Emmanuel N. Lazaridis, and David A. Williams. "Nonobese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) Mouse as a Model System to Study the Engraftment and Mobilization of Human Peripheral Blood Stem Cells." Blood 92, no. 7 (October 1, 1998): 2556–70. http://dx.doi.org/10.1182/blood.v92.7.2556.2556_2556_2570.
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Mobilized CD34+ cells from human peripheral blood (PB) are increasingly used for hematopoietic stem-cell transplantation. However, the mechanisms involved in the mobilization of human hematopoietic stem and progenitor cells are largely unknown. To study the mobilization of human progenitor cells in an experimental animal model in response to different treatment regimens, we injected intravenously a total of 92 immunodeficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with various numbers of granulocyte colony-stimulating factor (G-CSF) –mobilized CD34+ PB cells (ranging from 2 to 50 × 106cells per animal). Engraftment of human cells was detectable for up to 6.5 months after transplantation and, depending on the number of cells injected, reached as high as 96% in the bone marrow (BM), displaying an organ-specific maturation pattern of T- and B-lymphoid and myeloid cells. Among the different mobilization regimens tested, human clonogenic cells could be mobilized from the BM into the PB (P= .019) with a high or low dose of human G-CSF, alone or in combination with human stem-cell factor (SCF), with an average increase of 4.6-fold over control. Therefore, xenotransplantation of human cells in NOD/SCID mice will provide a basis to further study the mechanisms of mobilization and the biology of the mobilized primitive human hematopoietic cell.
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Chihiro, Tsunoda, Sakamoto Teruhiko, Yamada Rieko, Otsuka Hiroko, Mitsumaru Akiyoshi, Kawashima Megumi, Watanabe Noriko, Fujibayashi Mariko, Aiba Motohiko, and Kato Hiroyuki. "A case of primitive neuroectodermal tumor associated with Acquired Immunodeficiency Syndrome." Progress of Digestive Endoscopy 74, no. 2 (2009): 110–11. http://dx.doi.org/10.11641/pde.74.2_110.
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Marandin, A., A. Katz, E. Oksenhendler, M. Tulliez, F. Picard, W. Vainchenker, and F. Louache. "Loss of primitive hematopoietic progenitors in patients with human immunodeficiency virus infection." Blood 88, no. 12 (December 15, 1996): 4568–78. http://dx.doi.org/10.1182/blood.v88.12.4568.bloodjournal88124568.
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A number of hematologic abnormalities, including cytopenias, have been observed in patients with human immunodeficiency virus (HIV) infection. To elucidate their mechanisms, primitive cells from bone marrow aspirates of 21 patients with HIV-1 infection were quantitated by flow cytometry. The mean percentage of CD34+ cells is not significantly altered in HIV-1-infected patients in comparison with HIV-1- seronegative controls. In contrast, two- and three-color immunofluorescence analysis showed that in all HIV-1 samples, most CD34+ cells coexpressed the CD38 antigen. The proportion of HIV-1- derived CD34+ cells that did not express the CD38 antigen was significantly lower (HIV-1+: mean, 1.73%; controls: mean, 14%; P < .0005) than in controls. Moreover, of Thy-1+ cells, the proportion of CD34+ cells was twofold lower in HIV-1-infected patients (HIV-1+: mean, 12%; controls, 25%, P < .0005), which suggests that phenotypically primitive cells are depleted in HIV-1 infection. In vitro functional analysis in long-term cultures of sorted CD34+ cells from seven HIV-1 patients showed that CD34+ cells from HIV-1 patients generated much fewer colonies both in the nonadherent and adherent layers than CD34+ cells from controls after 5 weeks of culture (10-fold and four-fold less, respectively). Precise long-term culture initiating cell (LTC-IC) frequency in the CD34+ cell population was determined in three patients by limiting dilution and was markedly decreased in comparison to that of normal controls (from twofold to > sevenfold decreased). To determine if primitive cells were infected by HIV-1, both methylcellulose colonies generated from long-term culture of CD34+ cells and various CD34+ cell fractions purified by flow cytometry were evaluated for the presence of HIV-1 by polymerase chain reaction (PCR). Progeny from long-term culture was HIV-1-negative in three samples. In addition, using a sensitive PCR technique, the HIV-1 genome could not be detected in CD34+, CD34+/CD38-, and CD34+/CD4+ cells. These data show that hematologic disorders in HIV disease may be the consequence of a deficit of primitive cells. However, direct infection of these cells by HIV-1 does not seem to be responsible for this defect.
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Terpstra, W., RE Ploemacher, A. Prins, K. van Lom, K. Pouwels, AW Wognum, G. Wagemaker, B. Lowenberg, and JJ Wielenga. "Fluorouracil selectively spares acute myeloid leukemia cells with long- term growth abilities in immunodeficient mice and in culture." Blood 88, no. 6 (September 15, 1996): 1944–50. http://dx.doi.org/10.1182/blood.v88.6.1944.bloodjournal8861944.
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A subset of leukemic cells is assumed to maintain long-term growth of acute myeloid leukemia (AML) in vivo. Characterization of these AML progenitor cells may further define growth properties of human leukemia. In vitro incubations with 5-fluorouracil (5-FU) have been used for enrichment of normal primitive hematopoietic stem cells. By analogy to normal hematopoiesis, it was hypothesized that primitive leukemic stem cells might be kinetically more inactive than colony- forming cells (colony-forming units-AML [CFU-AML]). To examine this hypothesis, conditions were established for incubation with 5-FU that eliminated all CFU-AML. These conditions selected a 5-FU-resistant AML fraction that was evaluated for its capacity for long-term growth by transplantation into mice with severe combined immunodeficiency (SCID) and long-term culture in the quantitative cobblestone area-forming cell (CAFC) assay. Transplantation of the 5-FU-resistant fraction of four cases of AML into SCID mice resulted in growth of AML. Whereas no CFU- AML survived, 31% to 82% of primitive (week-6) CAFC were recovered from the 5-FU-treated cells. Hematopoietic cells proliferating in the CAFC assay were shown to be leukemic by cytologic, cytogenetic, or molecular analysis. The reduction of AML growth as determined by outgrowth of AML in SCID mice was in the same order of magnitude as the primitive (week- 6) CAFC reduction. This indicates that both assays measure closely related cell populations and that the CAFC assay can be used to study long-term growth of AML. These results show a hierarchy of AML cells that includes 5-FU-resistant progenitors. These cells are characterized as primitive (week-6) CAFC and as leukemia-initiating cells in SCID mice.
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Karanu, Frances N., Barbara Murdoch, Lisa Gallacher, Dongmei M. Wu, Masahide Koremoto, Seiji Sakano, and Mickie Bhatia. "The Notch Ligand Jagged-1 Represents a Novel Growth Factor of Human Hematopoietic Stem Cells." Journal of Experimental Medicine 192, no. 9 (November 6, 2000): 1365–72. http://dx.doi.org/10.1084/jem.192.9.1365.
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The Notch ligand, Jagged-1, plays an essential role in tissue formation during embryonic development of primitive organisms. However, little is known regarding the role of Jagged-1 in the regulation of tissue-specific stem cells or its function in humans. Here, we show that uncommitted human hematopoietic cells and cells that comprise the putative blood stem cell microenvironment express Jagged-1 and the Notch receptors. Addition of a soluble form of human Jagged-1 to cultures of purified primitive human blood cells had modest effects in augmenting cytokine-induced proliferation of progenitors. However, intravenous transplantation of cultured cells into immunodeficient mice revealed that human (h)Jagged-1 induces the survival and expansion of human stem cells capable of pluripotent repopulating capacity. Our findings demonstrate that hJagged-1 represents a novel growth factor of human stem cells, thereby providing an opportunity for the clinical utility of Notch ligands in the expansion of primitive cells capable of hematopoietic reconstitution.
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Cashman, Johanne, Ian Clark-Lewis, Allen Eaves, and Connie Eaves. "Stromal-derived factor 1 inhibits the cycling of very primitive human hematopoietic cells in vitro and in NOD/SCID mice." Blood 99, no. 3 (February 1, 2002): 792–99. http://dx.doi.org/10.1182/blood.v99.3.792.
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Abstract Stromal-derived factor 1 (SDF-1) is a -CXC- chemokine that plays a critical role in embryonic and adult hematopoiesis, and its specific receptor, CXCR4, has been implicated in stem cell homing. In this study, it is shown that the addition of SDF-1 to long-term cultures (LTCs) of normal human marrow can selectively, reversibly, and specifically block the S-phase entry of primitive quiescent erythroid and granulopoietic colony-forming cells (CFCs) present in the adherent layer. Conversely, addition of anti–SDF-1 antibody or SDF-1(G2), a specific CXCR4 antagonist, to preactivated human LTCs prevented both types of primitive CFCs from re-entering a quiescent state, demonstrating that endogenous SDF-1 contributes to the control of primitive CFC proliferation in the LTC system. Interestingly, SDF-1 failed to arrest the proliferation of primitive chronic myeloid leukemia CFCs in the adherent layer of LTCs containing normal marrow stromal cells. In vivo, injection of SDF-1 arrested the cycling of normal human LTC-initiating cells as well as primitive CFCs in the marrow of nonobese diabetic/severe combined immunodeficient mice engrafted with human cord blood cells. Conversely, injection of the antagonist, SDF-1(G2), reactivated the cycling of quiescent primitive human CFCs present in the marrow of mice engrafted with human marrow cells. These studies are the first to demonstrate a potential physiological role of SDF-1 in regulating the cell-cycle status of primitive hematopoietic cells and suggest that the deregulated cycling activity of primitive chronic myeloid leukemia (CML) cells is due to the BCR-ABL–mediated disruption of a pathway shared by multiple chemokine receptors.
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Milhem, Mohammed, Nadim Mahmud, Donald Lavelle, Hiroto Araki, Joseph DeSimone, Yogen Saunthararajah, and Ronald Hoffman. "Modification of hematopoietic stem cell fate by 5aza 2′deoxycytidine and trichostatin A." Blood 103, no. 11 (June 1, 2004): 4102–10. http://dx.doi.org/10.1182/blood-2003-07-2431.
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Abstract Efforts to change the fate of human hematopoietic stem cells (HSCs) and progenitor cells (HPCs) in vitro have met with limited success. We hypothesized that previously utilized in vitro conditions might result in silencing of genes required for the maintenance of primitive HSCs/HPCs. DNA methylation and histone deacetylation are components of an epigenetic program that regulates gene expression. Using pharmacologic agents in vitro that might possibly interfere with DNA methylation and histone deacetylation, we attempted to maintain and expand cells with phenotypic and functional characteristics of primitive HSCs/HPCs. Human marrow CD34+ cells were exposed to a cytokine cocktail favoring differentiation in combination with 5aza 2′deoxycytidine (5azaD) and trichostatin A (TSA), resulting in a significant expansion of a subset of CD34+ cells that possessed phenotypic properties as well as the proliferative potential characteristic of primitive HSCs/HPCs. In addition, 5azaD- and TSA-pretreated cells but not the CD34+ cells exposed to cytokines alone retained the ability to repopulate immunodeficient mice. Our findings demonstrate that 5azaD and TSA can be used to alter the fate of primitive HSCs/HPCs during in vitro culture.
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Kambe, Naotomo, Hidefumi Hiramatsu, Mika Shimonaka, Hisanori Fujino, Ryuta Nishikomori, Toshio Heike, Mamoru Ito, et al. "Development of both human connective tissue-type and mucosal-type mast cells in mice from hematopoietic stem cells with identical distribution pattern to human body." Blood 103, no. 3 (February 1, 2004): 860–67. http://dx.doi.org/10.1182/blood-2003-04-1160.
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Abstract The transplantation of primitive human cells into sublethally irradiated immune-deficient mice is the well-established in vivo system for the investigation of human hematopoietic stem cell function. Although mast cells are the progeny of hematopoietic stem cells, human mast cell development in mice that underwent human hematopoietic stem cell transplantation has not been reported. Here we report on human mast cell development after xenotransplantation of human hematopoietic stem cells into nonobese diabetic severe combined immunodeficient \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{NOD{/}SCID}){/}{\gamma}_{\mathrm{c}}^{null}\) \end{document} (NOG) mice with severe combined immunodeficiency and interleukin 2 (IL-2) receptor γ-chain allelic mutation. Supported by the murine environment, human mast cell clusters developed in mouse dermis, but they required more time than other forms of human cell reconstitution. In lung and gastric tract, mucosal-type mast cells containing tryptase but lacking chymase located on gastric mucosa and in alveoli, whereas connective tissue-type mast cells containing both tryptase and chymase located on gastric submucosa and around major airways, as in the human body. Mast cell development was also observed in lymph nodes, spleen, and peritoneal cavity but not in the peripheral blood. Xenotransplantation of human hematopoietic stem cells into NOG mice can be expected to result in a highly effective model for the investigation of human mast cell development and function in vivo.
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Woods, Niels-Bjarne, Cecilia Fahlman, Hanna Mikkola, Isao Hamaguchi, Karin Olsson, Romain Zufferey, Sten Eirik Jacobsen, Didier Trono, and Stefan Karlsson. "Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells." Blood 96, no. 12 (December 1, 2000): 3725–33. http://dx.doi.org/10.1182/blood.v96.12.3725.
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Abstract The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)–derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34+cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34+ cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34+population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400 000 cord blood CD34+ cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP+ lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells.
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Woods, Niels-Bjarne, Cecilia Fahlman, Hanna Mikkola, Isao Hamaguchi, Karin Olsson, Romain Zufferey, Sten Eirik Jacobsen, Didier Trono, and Stefan Karlsson. "Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells." Blood 96, no. 12 (December 1, 2000): 3725–33. http://dx.doi.org/10.1182/blood.v96.12.3725.h8003725_3725_3733.
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The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)–derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34+cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34+ cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34+population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400 000 cord blood CD34+ cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP+ lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells.
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Bhatia, Mickie, Dominique Bonnet, Ursula Kapp, Jean C. Y. Wang, Barbara Murdoch, and John E. Dick. "Quantitative Analysis Reveals Expansion of Human Hematopoietic Repopulating Cells After Short-term Ex Vivo Culture." Journal of Experimental Medicine 186, no. 4 (August 18, 1997): 619–24. http://dx.doi.org/10.1084/jem.186.4.619.
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Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38− cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38− cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.
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Kollet, Orit, Asaf Spiegel, Amnon Peled, Isabelle Petit, Tamara Byk, Rami Hershkoviz, Esther Guetta, Gad Barkai, Arnon Nagler, and Tsvee Lapidot. "Rapid and efficient homing of human CD34+CD38−/lowCXCR4+stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice." Blood 97, no. 10 (May 15, 2001): 3283–91. http://dx.doi.org/10.1182/blood.v97.10.3283.
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Abstract Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34+CD38−/lowCXCR4+ cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34−CD38−/lowLin− cells were not detected. Moreover, whereas freshly isolated CD34+CD38+/high cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34+CD38+ cells. Homing of enriched human CD34+ cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34+CD38−/lowCXCR4+cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34+ cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Gαiproteins, inhibited SDF-1–mediated in vitro transwell migration but not adhesion or in vivo homing of CD34+ cells. Homing of human CD34+ cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34+CD38−/lowCXCR4+cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.
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Mattapallil, Joseph J., Zeljka Smit-McBride, and Satya Dandekar. "Gastrointestinal Epithelium Is an Early Extrathymic Site for Increased Prevalence of CD34+ Progenitor Cells in Contrast to the Thymus during Primary Simian Immunodeficiency Virus Infection." Journal of Virology 73, no. 5 (May 1, 1999): 4518–23. http://dx.doi.org/10.1128/jvi.73.5.4518-4523.1999.
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ABSTRACT The objective of this study was to determine the effects of primary simian immunodeficiency virus (SIV) infection on the prevalence and phenotype of progenitor cells present in the gastrointestinal epithelia of SIV-infected rhesus macaques, a primate model for human immunodeficiency virus pathogenesis. The gastrointestinal epithelium was residence to progenitor cells expressing CD34 antigen, a subset of which also coexpressed Thy-1 and c-kit receptors, suggesting that the CD34+ population in the intestine comprised a subpopulation of primitive precursors. Following experimental SIVmac251 infection, an early increase in the proportions of CD34+ Thy-1+ and CD34+c-kit + progenitor cells was observed in the gastrointestinal epithelium. In contrast, the proportion of CD34+ cells in the thymus declined during primary SIV infection, which was characterized by a decrease in the frequency of CD34+ Thy-1+ progenitor cells. A severe depletion in the frequency of CD4-committed CD34+progenitors was observed in the gastrointestinal epithelium 2 weeks after SIV infection which persisted even 4 weeks after infection. A coincident increase in the frequency of CD8- committed CD34+ progenitor cells was observed during primary SIV infection. These results indicate that in contrast to the primary lymphoid organs such as the thymus, the gastrointestinal epithelium may be an early extrathymic site for the increased prevalence of both primitive and committed CD34+ progenitor cells. The gastrointestinal epithelium may potentially play an important role in maintaining T-cell homeostasis in the intestinal mucosa during primary SIV infection.
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Segovia, José C., Guillermo Guenechea, Jesús M. Gallego, José M. Almendral, and Juan A. Bueren. "Parvovirus Infection Suppresses Long-Term Repopulating Hematopoietic Stem Cells." Journal of Virology 77, no. 15 (August 1, 2003): 8495–503. http://dx.doi.org/10.1128/jvi.77.15.8495-8503.2003.
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ABSTRACT The functional disturbance of self-renewing and multipotent hematopoietic stem cells (HSCs) in viral diseases is poorly understood. In this report, we have assessed the susceptibility of mouse HSCs to strain i of the autonomous parvovirus minute virus of mice (MVMi) in vitro and during persistent infection of an immunodeficient host. Purified 5FUr Lin− Sca-1+ primitive hematopoietic precursors were permissive for MVMi genome replication and the expression of viral gene products. The lymphoid and myeloid repopulating capacity of bone marrow (BM) cells was significantly impaired after in vitro infection, although the degree of functional effect proportionally decreased with the posttransplantation time. This indicated that MVMi targets the heterogeneous compartment of repopulating cells with differential affinity and suggests that the virus may persist in some primitive HSCs in the quiescent stage, killing those eventually recruited for proliferative activity. Immunodeficient SCID mice oronasally infected with MVMi were cured of the characteristic virus-induced lethal leukopenia by transplantation of immunocompetent BM grafts. However, two double-stranded viral DNA species, probably uncommon replicative intermediates, remained in the marrow of every transplanted mouse months after infectious virus clearance. Genetic analysis of the rescued mice showed that the infection ensured a stable engraftment of donor hematopoiesis by markedly depleting the pool of endogenous HSCs. The MVMi-induced suppression of HSC functions illustrates the accessibility of this compartment to infection during a natural viral hematological disease. These results may provide clues to understanding delayed hematopoietic syndromes associated with persistent viral infections and to prospective gene delivery to HSCs in vivo.
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Moritz, T., D. C. Keller, and D. A. Williams. "Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease." Journal of Experimental Medicine 178, no. 2 (August 1, 1993): 529–36. http://dx.doi.org/10.1084/jem.178.2.529.
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Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.
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Srour, Edward F., Xia Tong, Ki Woong Sung, P. Artur Plett, Susan Rice, Joanne Daggy, Constantin T. Yiannoutsos, Rafat Abonour, and Christie M. Orschell. "Modulation of in vitro proliferation kinetics and primitive hematopoietic potential of individual human CD34+CD38–/lo cells in G0." Blood 105, no. 8 (April 15, 2005): 3109–16. http://dx.doi.org/10.1182/blood-2004-05-1773.
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AbstractWhether cytokines can modulate the fate of primitive hematopoietic progenitor cells (HPCs) through successive in vitro cell divisions has not been established. Single human marrow CD34+CD38–/lo cells in the G0 phase of cell cycle were cultured under 7 different cytokine combinations, monitored for proliferation on days 3, 5, and 7, then assayed for long-term culture-initiating cell (LTC-IC) function on day 7. LTC-IC function was then retrospectively correlated with prior number of in vitro cell divisions to determine whether maintenance of LTC-IC function after in vitro cell division is dependent on cytokine exposure. In the presence of proliferation progression signals, initial cell division was independent of cytokine stimulation, suggesting that entry of primitive HPCs into the cell cycle is a stochastic property. However, kinetics of proliferation beyond day 3 and maintenance of LTC-IC function were sensitive to cytokine stimulation, such that LTC-IC underwent an initial long cell cycle, followed by more synchronized shorter cycles varying in length depending on the cytokine combination. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) transplantation studies revealed analogous results to those obtained with LTC-ICs. These data suggest that although exit from quiescence and commitment to proliferation might be stochastic, kinetics of proliferation, and possibly fate of primitive HPCs, might be modulated by extrinsic factors.
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Hess, David A., Todd E. Meyerrose, Louisa Wirthlin, Timothy P. Craft, Phillip E. Herrbrich, Michael H. Creer, and Jan A. Nolta. "Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity." Blood 104, no. 6 (September 15, 2004): 1648–55. http://dx.doi.org/10.1182/blood-2004-02-0448.
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Abstract Human hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture, purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin-) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDHhiLin- cells contained 22.6% ± 3.0% of the Lin- population and highly coexpressed primitive HSC phenotypes (CD34+ CD38- and CD34+CD133+). In vitro hematopoietic progenitor function was enriched in the ALDHhiLin- population, compared with ALDHloLin- cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDHhiLin- cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID β2 microglobulin (β2M) null models demonstrated that 10-fold greater numbers of ALDHhi-Lin- cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID β2M null mouse, suggesting that the ALDHhiLin- population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype. (Blood. 2004;104:1648-1655)
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Muguruma, Yukari, Takashi Yahata, Hiroko Miyatake, Tadayuki Sato, Tomoko Uno, Jobu Itoh, Shunichi Kato, Mamoru Ito, Tomomitsu Hotta, and Kiyoshi Ando. "Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment." Blood 107, no. 5 (March 1, 2006): 1878–87. http://dx.doi.org/10.1182/blood-2005-06-2211.
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Hematopoiesis is maintained by specific interactions between both hematopoietic and nonhematopoietic cells. Whereas hematopoietic stem cells (HSCs) have been extensively studied both in vitro and in vivo, little is known about the in vivo characteristics of stem cells of the nonhematopoietic component, known as mesenchymal stem cells (MSCs). Here we have visualized and characterized human MSCs in vivo following intramedullary transplantation of enhanced green fluorescent protein-marked human MSCs (eGFP-MSCs) into the bone marrow (BM) of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Between 4 to 10 weeks after transplantation, eGFP-MSCs that engrafted in murine BM integrated into the hematopoietic microenvironment (HME) of the host mouse. They differentiated into pericytes, myofibroblasts, BM stromal cells, osteocytes in bone, bone-lining osteoblasts, and endothelial cells, which constituted the functional components of the BM HME. The presence of human MSCs in murine BM resulted in an increase in functionally and phenotypically primitive human hematopoietic cells. Human MSC-derived cells that reconstituted the HME appeared to contribute to the maintenance of human hematopoiesis by actively interacting with primitive human hematopoietic cells.
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Chadwick, Kristin, Farbod Shojaei, Lisa Gallacher, and Mickie Bhatia. "Smad7 alters cell fate decisions of human hematopoietic repopulating cells." Blood 105, no. 5 (March 1, 2005): 1905–15. http://dx.doi.org/10.1182/blood-2004-03-0881.
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AbstractIntracellular Smad proteins mediate signal transduction of the transforming growth factor-β (TGF-β) superfamily that play pleiotropic roles in hematopoietic development, suggesting that intracellular Smad proteins may play key roles in hematopoietic regulation. Although inhibitory Smad7, which negatively regulates TGF-β signaling, has been implicated in the development of mature hematopoietic cells, a role for Smad7 in regulating more primitive hematopoietic cells has yet to be examined. Here, Smad7 was overexpressed in primary human severe combined immunodeficient (SCID) repopulating cells (SRCs), representing a common myeloid/lymphoid precursor cell with the functional capacity to repopulate the bone marrow of nonobese diabetic (NOD)/SCID recipient mice. Retroviral transduction of Smad7 into human umbilical cord blood (CB)-SRCs caused a shift from lymphoid dominant engraftment toward increased myeloid contribution, and increased the myeloid-committed clonogenic progenitor frequency in reconstituted mice. Neither myeloid nor B-lymphoid lineage developmental stages were compromised by Smad7 overexpression, suggesting Smad7 regulates cell fate commitment decisions of myeloid/lymphoid precursors by augmenting myeloid differentiation at the expense of lymphoid commitment. In addition, global gene expression analysis using microarray was used to identify potential target genes regulated by Smad7 in primitive hematopoietic cells that may control this process. Our study demonstrates a novel and unexpected role for Smad7 in modulating the cell fate decisions of primary multipotent human repopulating cells and establishes a role for Smad7 in the development of primitive human hematopoietic cells.
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Cashman, Johanne D., Tsvee Lapidot, Jean C. Y. Wang, Monica Doedens, Leonard D. Shultz, Peter Lansdorp, John E. Dick, and Connie J. Eaves. "Kinetic Evidence of the Regeneration of Multilineage Hematopoiesis From Primitive Cells in Normal Human Bone Marrow Transplanted Into Immunodeficient Mice." Blood 89, no. 12 (June 15, 1997): 4307–16. http://dx.doi.org/10.1182/blood.v89.12.4307.
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Abstract Based on initial observations of human CD34+ Thy-1+ cells and long-term culture-initiating cells (LTC-IC) in the bone marrow of some sublethally irradiated severe combined immunodeficient (SCID) mice transplanted intravenously with normal human marrow cells, and the subsequent finding that the NOD/LtSz-scid/scid (NOD/SCID) mouse supports higher levels of human cell engraftment, we undertook a series of time course experiments to examine posttransplant changes in the number, tissue distribution, cycling activity, and in vivo differentiation pattern of various human hematopoietic progenitor cell populations in this latter mouse model. These studies showed typical rapid posttransplant recovery curves for human CD34− CD19+ (B-lineage) cells, CD34+ granulopoietic, erythroid, and multilineage colony-forming cells (CFC), LTC-IC, and CD34+ Thy-1+ cells from a small initial population representing <0.1% of the original transplant. The most primitive human cell populations reached maximum values at 5 weeks posttransplant, after which they declined. More mature cell types peaked after another 5 weeks and then declined. A 2-week course of thrice weekly injections of human Steel factor, interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (administered just before the mice were killed for analysis) did not alter the pace of regeneration of either primitive or mature human hematopoietic cells, or their predominantly granulopoietic and B-lymphoid pattern of differentiation, although a significant enhancing effect on the level of human cell engraftment sustained after 3 months was noted. Cycling studies showed the human CFC present at 4 to 5 weeks posttransplant to be rapidly proliferating even in mice not given human growth factors. However, by 10 weeks and thereafter, only quiescent human CFC were detected; interestingly, even in mice that were given the 2-week course of growth factor injections. These studies indicate the use of this model for future analysis of the properties and in vivo regulation of primitive human hematopoietic cells that possess in vivo repopulating ability.
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Tremont-Lukats, I. W., Gilda M. Teixeira, and Dimas E. Hernández. "Primitive reflexes in a case-control study of patients with advanced human immunodeficiency virus type 1." Journal of Neurology 246, no. 7 (July 22, 1999): 540–43. http://dx.doi.org/10.1007/s004150050400.
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Chaligné, Ronan, Chloé James, Carole Tonetti, Rodolphe Besancenot, Jean Pierre Le Couédic, Fanny Fava, Fréderic Mazurier, et al. "Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis." Blood 110, no. 10 (November 15, 2007): 3735–43. http://dx.doi.org/10.1182/blood-2007-05-089003.
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Abstract The MPL (W515L and W515K) mutations have been detected in granulocytes of patients suffering from certain types of primitive myelofibrosis (PMF). It is still unknown whether this molecular event is also present in lymphoid cells and therefore potentially at the hematopoietic stem cell (HSC) level. Toward this goal, we conducted MPL genotyping of mature myeloid and lymphoid cells and of lymphoid/myeloid progenitors isolated from PMF patients carrying the W515 mutations. We detected both MPL mutations in granulocytes, monocytes, and platelets as well as natural killer (NK) cells but not in T cells. B/NK/myeloid and/or NK/myeloid CD34+CD38−-derived clones were found to carry the mutations. Long-term reconstitution of MPL W515 CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice was successful for as long as 12 weeks after transplantation, indicating that MPL W515 mutations were present in HSCs. Moreover, the 2 MPL mutations induced a spontaneous megakaryocytic growth in culture with an overall normal response to thrombopoietin (TPO). In contrast, erythroid progenitors remained EPO dependent. These results demonstrate that in PMF, the MPL W515L or K mutation induces a spontaneous megakaryocyte (MK) differentiation and occurs in a multipotent HSCs.
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Cheung, Alice M. S., and Connie J. Eaves. "Human Cord Blood Cells with Rapid In Vivo Platelet Regenerating Activity in NOD/SCID-IL-2Rγc-/- Mice Show Variable Aldehyde Dehydrogenase Activity." Blood 114, no. 22 (November 20, 2009): 2429. http://dx.doi.org/10.1182/blood.v114.22.2429.2429.
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Abstract Abstract 2429 Poster Board II-406 Human cord blood (CB) has emerged as an attractive source of hematopoietic cells for patients lacking a suitable donor. However, marked delays in platelet and immune recovery pose significant challenges to the use of CB cells as transplants for either children or adult patients. These difficulties in the use of CB have been attributed to low absolute numbers of repopulating cells (RCs) in most CB units which is not readily overcome by simply combining multiple units. Xenotransplantation of human hematopoietic cells into highly immunodeficient sublethally irradiated NOD/SCID mice has proven to be a powerful approach to characterize different types of primitive human hematopoietic cells with repopulating potential. However, the residual NK activity intrinsic to NOD/SCID mice poses a significant barrier to the engraftment of intermediate types of repopulating human cells and also to the terminal stages of their differentiation, as shown by recent studies using more immunodeficient mice as hosts. Nevertheless, the cells responsible for early platelet recovery post-transplant and factors that regulate their activity remain largely unknown. To address this issue, we have developed a quantitative and sensitive assay for characterizing the phenotypes of human CB cells that regenerate mature platelets detectable in the blood of NOD/SCID-IL-2Rγc-/- mice 3-6 weeks post-transplant. Lineage marker-negative (Lin-) human CB cells were stained with Aldefluor and then those with low light side-scattering properties were further separated by FACS according to whether they displayed aldehyde dehydrogenase activity above (ALDH+) or below (ALDH-) that detected in the presence of an ALDH inhibitor. Assays of different pooled human CB preparations showed that the most primitive class of in vitro megakaryocyte (Mk) colony-forming cells and cells responsible for rapid human platelet production in NOD/SCID-IL-2Rγc-/- mice were variably and comparably distributed between the small ALDH+ and prevalent ALDH- fractions. From 3 experiments, the following values were obtained for the ALDH+ and ALDH- subsets, respectively; ALDH+ - mature CFU-Mk = 47.8±21.3% of the total Lin- fraction, intermediate CFU-Mk = 66.3±25.4%, primitive CFU-Mk = 75.5±14.0%, 3-week platelet-producing STRC = 63.8±12.3%, 6-week platelet-producing STRC = 81.5±5.8%; and ALDH- - mature CFU-Mk = 52.2±21.3% of the total Lin- fraction, intermediate CFU-Mk = 33.7±25.4%, primitive CFU-Mk = 24.5±14.0%, 3-week platelet-producing STRC = 36.2±12.3%, 6-week platelet-producing STRC = 18.5±5.8%. Limiting dilution assays revealed 1 in 830 Lin-ALDH+ CB cells to be a cell that can produce detectable platelets in vivo within 3 weeks (95% CI = 1 in 534 to 1 in 1290) but only 1 in 1996 (95% CI = 1 in 1226 to 1 in 3247) within 6 weeks. The present study demonstrates the feasibility of using NOD/SCID-IL-2Rγc-/- mice for the sensitive detection of human CB cells with in vivo platelet regenerating activity and suggests that these may be closely related to primitive cells with in vitro Mk clonogenic activity (>50 Mk per colony). Biologically important platelet progenitors may thus be heterogeneous with respect to ALDH+ activity. Disclosures: No relevant conflicts of interest to declare.
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Bennaceur-Griscelli, Annelise, Cristina Tourino, Brigitte Izac, William Vainchenker, and Laure Coulombel. "Murine Stromal Cells Counteract the Loss of Long-Term Culture-Initiating Cell Potential Induced by Cytokines in CD34+CD38low/neg Human Bone Marrow Cells." Blood 94, no. 2 (July 15, 1999): 529–38. http://dx.doi.org/10.1182/blood.v94.2.529.
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Abstract Evidence has been provided recently that shows that high concentrations of cytokines can fulfill functions previously attributed to stromal cells, such as promote the survival of, and led to a net increase in human primitive progenitors initiating long-term cultures in vitro (LTC-IC) or engrafting NOD-SCID (nonobese diabetic severe-combined immunodeficient) recipients in vivo. These data prompted us to re-evaluate whether stromal cells will further alter the properties of primitive progenitor cells exposed to cytokines. Single CD34+CD38low and CD38neg cells were incubated 10 days in serum-containing or serum-free medium in the presence or in the absence of murine marrow-derived stromal cells (MS-5). Recombinant human cytokines stem cell factor (SCF), pegylated-megakaryocyte growth and differentiation factor (PEG–MGDF), FLT3-L, Interleukin (IL)-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM–CSF) were systematically added at various concentrations (10 to 300 ng/mL). Cell proliferation and LTC-IC potential were evaluated in each clone after 10 days. A striking and consistent observation was the retention of a high LTC-IC potential in clones exposed to cytokines in the presence of stromal feeders, whereas clones exposed to cytokines alone in the absence of stromal feeders rapidly lost their LTC-IC potential as they proliferated. This was reflected both by the higher proportion of wells containing LTC-IC and by the high numbers of CFC produced after 5 weeks in clones grown with MS-5 during the first 10 days. We further showed by analyzing multiple replicates of a single clone at day 10 that MS-5 cells promoted a net increase in the LTC-IC compartment through self-renewal divisions. Interestingly, these primitive LTC-IC were equally distributed among small and large clones, as counted at day 10, indicating that active proliferation and loss of LTC-IC potential could be dissociated. These observations show that, in primitive cells, stromal cells counteract differentiation events triggered by cytokines and promoted self-renewal divisions. Furthermore, the almost identical distribution of the size of the clones with or without MS-5 suggests that proliferation and function of human primitive cells may be independently regulated by external signals, and that the former is primarily under the control of cytokines.
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Bennaceur-Griscelli, Annelise, Cristina Tourino, Brigitte Izac, William Vainchenker, and Laure Coulombel. "Murine Stromal Cells Counteract the Loss of Long-Term Culture-Initiating Cell Potential Induced by Cytokines in CD34+CD38low/neg Human Bone Marrow Cells." Blood 94, no. 2 (July 15, 1999): 529–38. http://dx.doi.org/10.1182/blood.v94.2.529.414k21_529_538.
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Evidence has been provided recently that shows that high concentrations of cytokines can fulfill functions previously attributed to stromal cells, such as promote the survival of, and led to a net increase in human primitive progenitors initiating long-term cultures in vitro (LTC-IC) or engrafting NOD-SCID (nonobese diabetic severe-combined immunodeficient) recipients in vivo. These data prompted us to re-evaluate whether stromal cells will further alter the properties of primitive progenitor cells exposed to cytokines. Single CD34+CD38low and CD38neg cells were incubated 10 days in serum-containing or serum-free medium in the presence or in the absence of murine marrow-derived stromal cells (MS-5). Recombinant human cytokines stem cell factor (SCF), pegylated-megakaryocyte growth and differentiation factor (PEG–MGDF), FLT3-L, Interleukin (IL)-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM–CSF) were systematically added at various concentrations (10 to 300 ng/mL). Cell proliferation and LTC-IC potential were evaluated in each clone after 10 days. A striking and consistent observation was the retention of a high LTC-IC potential in clones exposed to cytokines in the presence of stromal feeders, whereas clones exposed to cytokines alone in the absence of stromal feeders rapidly lost their LTC-IC potential as they proliferated. This was reflected both by the higher proportion of wells containing LTC-IC and by the high numbers of CFC produced after 5 weeks in clones grown with MS-5 during the first 10 days. We further showed by analyzing multiple replicates of a single clone at day 10 that MS-5 cells promoted a net increase in the LTC-IC compartment through self-renewal divisions. Interestingly, these primitive LTC-IC were equally distributed among small and large clones, as counted at day 10, indicating that active proliferation and loss of LTC-IC potential could be dissociated. These observations show that, in primitive cells, stromal cells counteract differentiation events triggered by cytokines and promoted self-renewal divisions. Furthermore, the almost identical distribution of the size of the clones with or without MS-5 suggests that proliferation and function of human primitive cells may be independently regulated by external signals, and that the former is primarily under the control of cytokines.
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Weichold, Frank F., Davide Zella, Oxana Barabitskaja, Jaroslaw P. Maciejewski, Daniel E. Dunn, Elaine M. Sloand, and Neal S. Young. "Neither Human Immunodeficiency Virus-1 (HIV-1) nor HIV-2 Infects Most-Primitive Human Hematopoietic Stem Cells as Assessed in Long-Term Bone Marrow Cultures." Blood 91, no. 3 (February 1, 1998): 907–15. http://dx.doi.org/10.1182/blood.v91.3.907.
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Abstract Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38−) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.
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Weichold, Frank F., Davide Zella, Oxana Barabitskaja, Jaroslaw P. Maciejewski, Daniel E. Dunn, Elaine M. Sloand, and Neal S. Young. "Neither Human Immunodeficiency Virus-1 (HIV-1) nor HIV-2 Infects Most-Primitive Human Hematopoietic Stem Cells as Assessed in Long-Term Bone Marrow Cultures." Blood 91, no. 3 (February 1, 1998): 907–15. http://dx.doi.org/10.1182/blood.v91.3.907.907_907_915.
Full textAbstract:
Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38−) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.
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Brun, Ann C. M., Jon Mar Björnsson, Mattias Magnusson, Nina Larsson, Per Leveén, Mats Ehinger, Eva Nilsson, and Stefan Karlsson. "Hoxb4-deficient mice undergo normal hematopoietic development but exhibit a mild proliferation defect in hematopoietic stem cells." Blood 103, no. 11 (June 1, 2004): 4126–33. http://dx.doi.org/10.1182/blood-2003-10-3557.
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Abstract Enforced expression of Hoxb4 dramatically increases the regeneration of murine hematopoietic stem cells (HSCs) after transplantation and enhances the repopulation ability of human severe combined immunodeficiency (SCID) repopulating cells. Therefore, we asked what physiologic role Hoxb4 has in hematopoiesis. A novel mouse model lacking the entire Hoxb4 gene exhibits significantly reduced cellularity in spleen and bone marrow (BM) and a subtle reduction in red blood cell counts and hemoglobin values. A mild reduction was observed in the numbers of primitive progenitors and stem cells in adult BM and fetal liver, whereas lineage distribution was normal. Although the cell cycle kinetics of primitive progenitors was normal during endogenous hematopoiesis, defects in proliferative responses of BM Lin- Sca1+ c-kit+ stem and progenitor cells were observed in culture and in vivo after the transplantation of BM and fetal liver HSCs. Quantitative analysis of mRNA from fetal liver revealed that a deficiency of Hoxb4 alone changed the expression levels of several other Hox genes and of genes involved in cell cycle regulation. In summary, the deficiency of Hoxb4 leads to hypocellularity in hematopoietic organs and impaired proliferative capacity. However, Hoxb4 is not required for the generation of HSCs or the maintenance of steady state hematopoiesis.
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Reynaud, Damien, Emmanuel Ravet, Monique Titeux, Frédéric Mazurier, Laurent Rénia, Anne Dubart-Kupperschmitt, Paul-Henri Roméo, and Françoise Pflumio. "SCL/TAL1 expression level regulates human hematopoietic stem cell self-renewal and engraftment." Blood 106, no. 7 (October 1, 2005): 2318–28. http://dx.doi.org/10.1182/blood-2005-02-0557.
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AbstractThe fate of hematopoietic stem cells (HSCs) is regulated through a combinatorial action of proteins that determine their self-renewal and/or their commitment to differentiation. Stem cell leukemia/T-cell acute lymphoblastic leukemia 1 (SCL/TAL1), a basic helix-loop-helix (bHLH) transcription factor, plays key roles in controlling the development of primitive and definitive hematopoiesis during mouse development but its function in adult HSCs is still a matter of debate. We report here that the lentiviral-mediated enforced expression of TAL1 in human CD34+ cells marginally affects in vitro the differentiation of committed progenitors, whereas in vivo the repopulation capacity of the long-term SCID (severe combined immunodeficient) mouse–repopulating cells (LT-SRCs) is enhanced. As a consequence, the production of SRC-derived multipotent progenitors as well as erythroid- and myeloid-differentiated cells is increased. Looking at the lymphoid compartment, constitutive TAL1-enforced expression impairs B- but not T-cell differentiation. Expression of a mutant TAL1 protein that cannot bind DNA specifically impairs human LT-SRC amplification, indicating a DNA-binding dependent effect of TAL1 on primitive cell populations. These results indicate that TAL1 expression level regulates immature human hematopoietic cell self-renewal and that this regulation requires TAL1 DNA-binding activity.
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Tremont-Lukats, I. W., R. Serbanescu, G. M. Teixeira, E. Iriza, D. E. Hernández, and C. Schneider. "Multivariate analysis of primitive reflexes in patients with human immunodeficiency virus type-1 infection and neurocognitive dysfunction." Italian Journal of Neurological Sciences 20, no. 1 (April 12, 1999): 17–22. http://dx.doi.org/10.1007/s100720050005.
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Herzog, W. R., R. Meade, A. Pettinicchi, W. Ptak, and P. W. Askenase. "Nude mice produce a T cell-derived antigen-binding factor that mediates the early component of delayed-type hypersensitivity." Journal of Immunology 142, no. 6 (March 15, 1989): 1803–12. http://dx.doi.org/10.4049/jimmunol.142.6.1803.
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Abstract The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is caused by the sequential action of two different T cells. An early-acting, DTH-initiating T cell produces an Ag-specific T cell factor, that is analogous to IgE antibody and initiates DTH by sensitizing the local tissues for release of the vasoactive amine serotonin. In picryl chloride or oxazolone contact sensitivity, this T cell factor is Ag-specific, but MHC unrestricted. We, therefore, hypothesized that DTH-initiating T cells are primitive T cells with Ag receptors that can bind Ag without MHC restriction. In order to characterize the origin of this DTH-initiating T cell and the conditions that are necessary for its development, we contact-sensitized various strains of immunodeficient mice. Surprisingly, we found that the early phase of DTH was present in athymic nude mice. In contrast, the early component of DTH was absent in mice with severe combined immunodeficiency. These mice lack T and B cells, but have NK cells. These findings suggested that the early component of DTH was not caused by NK cells, and was caused by cells belonging to a lineage from a rearranging gene family. The early component of DTH in nude mice was Ag specific, was caused by MHC unrestricted Thy-1+ T cells, and was mediated by Ag-binding, Ag-specific T cell factors. We found that DTH-initiating, T cell-derived, Ag-binding molecules from nude mice and normal CBA/J mice had the same functional properties. The early component of DTH was elicited in two different systems (contact sensitivity and SRBC-specific DTH) in two strains of nude mice (BALB/c athymic nudes and CByB6F1/J-nu) from two different suppliers, but not in BALB/c and athymic nudes from a third supplier. From these findings we concluded that DTH-initiating T cells, which produce IgE-like Ag-specific T cell factors, are present in some strains of athymic nude mice and thus are relatively thymic independent T cells.
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Rissone, Alberto, Jaya Jagadeesh, Karen Simon, Kevin Bishop, Raman B. Sood, and Fabio Candotti. "AK2 Deficiency In Zebrafish Recapitulates Human Reticular Dysgenesis, An Autosomal Recessive Form Of Severe Combined Immunodeficiency." Blood 122, no. 21 (November 15, 2013): 2416. http://dx.doi.org/10.1182/blood.v122.21.2416.2416.
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Abstract The adenylate kinase (AK) gene family consists of 7 different members that contribute to energy cell metabolism by converting ATP+AMP to 2ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues. Mutations in the AK2 gene are responsible for reticular dysgenesis (RD), an autosomal recessive form of severe combined immunodeficiency (SCID). RD is characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation and it represents less than 2% of total SCID. Affected children succumb to overwhelming infections early in life unless their immune system is successfully restored with allogeneic hematopoietic stem cells transplant (HSCT). The mechanisms underlying the pathophysiology of RD remain unclear. The phenotype of AK2 deficient animals has never been reported in the literature, but murine lines carrying homozygous inactivating retroviral insertions are embryonically lethal (our personal observations). We used the zebrafish model to perform a comprehensive study of the effects of AK2 deficiency using Morpholino oligomers injections and two different kinds of AK2 mutants (a ENU-induced T371C/L124P missense mutant and two null mutant lines generated using zinc-finger nuclease technology). In situ hybridization analyses of AK2-deficient embryos indicated that only erythroid development was affected during primitive hematopoiesis. Conversely, during definitive hematopoiesis, the loss of function of AK2 resulted in abnormalities distributed along all hematopoietic lineages suggesting an impairment of hematopoietic stem cell (HSC) development. Moreover, we observed that the AK2 deficiency induced oxidative stress and consequent apoptosis in both primitive erythroid cells and definitive HSCs. Importantly, antioxidant treatment of AK2 mutant embryos rescued the hematopoietic phenotypes as indicated by the recovered expression of HSC and lymphoid markers (such as c-myb and rag1). Overall, our data indicate that zebrafish represents a good model for studying the molecular mechanisms involved in RD and testing of new therapeutic interventions. To date, our mutant lines remain the only animal model of this rare and lethal human disease. Disclosures: No relevant conflicts of interest to declare.
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Nguyen, Thu Yen Thi, Alessandra Camboni, Rossella Masciangelo, Jacques Donnez, and Marie-Madeleine Dolmans. "Is Ovarian Tissue Transplantation Safe in Patients with Central Nervous System Primitive Neuroectodermal Tumors?" Journal of Clinical Medicine 9, no. 12 (December 18, 2020): 4101. http://dx.doi.org/10.3390/jcm9124101.
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The risk of reseeding malignancy harbored in cryopreserved and transplanted ovarian tissue has been a source of concern. This study aimed to determine the potential relationship between frozen–thawed ovarian tissue transplantation and primary cancer recurrence. Three patients with cerebral primitive neuroectodermal tumors (PNET) were included in this study. One woman gave birth to three healthy babies following reimplantation of her cryopreserved ovarian tissue, but subsequently died due to cancer relapse six years after ovarian tissue transplantation. The second subject died from progressive cancer, while the third is still alive and awaiting reimplantation of her ovarian tissue in due course. Frozen ovarian cortex from all three patients was analyzed and xenotransplanted to immunodeficient mice for five months. Main outcomes were the presence of cancer cells in the thawed and xenografted ovarian tissue at histology, immunostaining (expression of neuron-specific enolase and glial fibrillary acidic protein (GFAP)), and reverse-transcription droplet digital polymerase chain reaction (RT-ddPCR) (levels of enolase 2 and GFAP). In conclusion, no malignant cells were detected in ovarian tissue from patients with PNET, even in those who experienced recurrence of the disease, meaning that the risk of reseeding cancer cells with ovarian tissue transplantation in these patients can be considered low.
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Gallacher, Lisa, Barbara Murdoch, Dongmei Wu, Francis Karanu, Fraser Fellows, and Mickie Bhatia. "Identification of novel circulating human embryonic blood stem cells." Blood 96, no. 5 (September 1, 2000): 1740–47. http://dx.doi.org/10.1182/blood.v96.5.1740.
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Abstract Using murine models, primitive hematopoietic cells capable of repopulation have been shown to reside in various anatomic locations, including the aortic gonad mesonephros, fetal liver, and bone marrow. These sites are thought to be seeded by stem cells migrating through fetal circulation and would serve as ideal targets for in utero cellular therapy. In humans, however, it is unknown whether similar stem cells exist. Here, we identify circulating hematopoeitic cells present during human in utero development that are capable of multilineage repopulation in immunodeficient NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Using limiting dilution analysis, the frequency of these fetal stem cells was found to be 1 in 3.2 × 105, illustrating a 3- and 22-fold enrichment compared with full-term human cord blood and circulating adult mobilized–peripheral blood, respectively. Comparison of in vivo differentiation and proliferative capacity demonstrated that circulating fetal stem cells are intrinsically distinct from hematopoietic stem cells found later in human development and those derived from the fetal liver or fetal bone marrow compartment at equivalent gestation. Taken together, these studies demonstrate the existence of unique circulating stem cells in early human embryonic development that provide a novel and previously unexplored source of pluripotent stem cell targets for cellular and gene-based fetal therapies.
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Gallacher, Lisa, Barbara Murdoch, Dongmei Wu, Francis Karanu, Fraser Fellows, and Mickie Bhatia. "Identification of novel circulating human embryonic blood stem cells." Blood 96, no. 5 (September 1, 2000): 1740–47. http://dx.doi.org/10.1182/blood.v96.5.1740.h8001740_1740_1747.
Full textAbstract:
Using murine models, primitive hematopoietic cells capable of repopulation have been shown to reside in various anatomic locations, including the aortic gonad mesonephros, fetal liver, and bone marrow. These sites are thought to be seeded by stem cells migrating through fetal circulation and would serve as ideal targets for in utero cellular therapy. In humans, however, it is unknown whether similar stem cells exist. Here, we identify circulating hematopoeitic cells present during human in utero development that are capable of multilineage repopulation in immunodeficient NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Using limiting dilution analysis, the frequency of these fetal stem cells was found to be 1 in 3.2 × 105, illustrating a 3- and 22-fold enrichment compared with full-term human cord blood and circulating adult mobilized–peripheral blood, respectively. Comparison of in vivo differentiation and proliferative capacity demonstrated that circulating fetal stem cells are intrinsically distinct from hematopoietic stem cells found later in human development and those derived from the fetal liver or fetal bone marrow compartment at equivalent gestation. Taken together, these studies demonstrate the existence of unique circulating stem cells in early human embryonic development that provide a novel and previously unexplored source of pluripotent stem cell targets for cellular and gene-based fetal therapies.
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Yoshimoto, Momoko, Prashanth Porayette, Nicole L. Glosson, Simon J. Conway, Nadia Carlesso, Angelo A. Cardoso, Mark H. Kaplan, and Mervin C. Yoder. "Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence." Blood 119, no. 24 (June 14, 2012): 5706–14. http://dx.doi.org/10.1182/blood-2011-12-397489.
Full textAbstract:
Abstract The extra-embryonic yolk sac (YS) is the first hematopoietic site in the mouse embryo and is thought to generate only primitive erythroid and myeloerythroid progenitor cells before definitive HSC emergence within the embryo on E10.5. Here, we have shown the existence of T cell–restricted progenitors in the E9.5 YS that directly engraft in recipient immunodeficient mice. T-cell progenitors were also produced in vitro from both YS and para-aortic splanchnopleura hemogenic endothelial cells, and these T-cell progenitors repopulated the thymus and differentiated into mature T-cell subsets in vivo on transplantation. Our data confirm that the YS produces T-lineage–restricted progenitors that are available to colonize the thymus and provide new insight into the YS as a definitive hematopoietic site in the mouse embryo.
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Rissone, Alberto, Jaya Jagadeesh, Simon Karen, Kevin Bishop, Trevor Blake, Raman B. Sood, and Fabio Candotti. "Adenylate Kinase 2 Regulates Zebrafish Primitive and Definitive Hematopoiesis." Blood 120, no. 21 (November 16, 2012): 1208. http://dx.doi.org/10.1182/blood.v120.21.1208.1208.
Full textAbstract:
Abstract Abstract 1208 Introduction: The adenylate kinase (AK) gene family consists of 7 different members that contribute to energy cell metabolism by converting ATP + AMP to 2 molecules of ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues and are generally involved in a broad range of cellular functions. Among AKs, AK2 has unique features such as its location in the mitochondrial intermembrane space and critical role in human lymphopoiesis and granulopoiesis. Indeed, mutations of the AK2 gene cause reticular dysgenesis (RD), an autosomal recessive form of severe combined immunodeficiency (SCID) characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation. The mechanisms underlying the pathophysiology of RD remain unclear. The phenotype of AK2 deficient animals has not been reported in the literature, but murine lines carrying homozygous inactivating retroviral insertions are embryonically lethal (our personal observations). Objectives: To study the role of AK2 in hematopoietic system development and define the effects of AK2 deficiency, we set out to generate a zebrafish model of RD. Methods: We injected zebrafish embryos with morpholino oligomers specific for the two zebrafish AK2 isoforms and analyzed the serial expression pattern of several hematopoietic markers in developing AK2 morphants. To confirm our observations in AK2 knockdown embryos, we screened a zebrafish DNA library of ENU-induced mutations and recovered a mutant fish line carrying a T371C/L124P missense mutation within the exon 4 of AK2 gene that is predicted to be deleterious for protein stability and function. Results: The downregulation of zebrafish AK2 expression phenocopied the human disease and resulted in strong reduction of developing lymphocytes. In addition, in situ hybridization for GATA1, alpha-globin 1, L-plastin and Odianisidine staining indicated that erythroid development was affected in AK2 morphants during primitive hematopoiesis, while myeloid development was conserved. Furthermore, in situ hybridization studies of the expression of markers of zebrafish definitive hematopoiesis showed abnormalities distributed among all hematopoietic lineages suggesting a broad role of AK2 in zebrafish hematopoiesis. Importantly, the ENU-induced Ak2 mutant recapitulated all the primitive and definitive hematopoietic phenotypes observed in AK2 morphants. Finally, preliminary data suggest that AK2 deficiency (both in morphant and mutant embryos) induces an increased level of reactive oxygen species (ROS) triggering oxidative stress and consequent apoptosis in hematopoietic progenitor cells. Conclusions: Our data provide new insights into the AK2 function and indicate that zebrafish represents a good model for studying the molecular mechanisms involved in RD. To date, our mutant line represents the first example of animal model of this rare and unique human disease. Disclosures: No relevant conflicts of interest to declare.
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Renelt, Sebastian, Patrizia Schult-Dietrich, Hanna-Mari Baldauf, Stefan Stein, Gerrit Kann, Markus Bickel, Ulrikke Kielland-Kaisen, et al. "HIV-1 Infection of Long-Lived Hematopoietic Precursors In Vitro and In Vivo." Cells 11, no. 19 (September 23, 2022): 2968. http://dx.doi.org/10.3390/cells11192968.
Full textAbstract:
Latent reservoirs in human-immunodeficiency-virus-1 (HIV-1)-infected individuals represent a major obstacle in finding a cure for HIV-1. Hematopoietic stem and progenitor cells (HSPCs) have been described as potential HIV-1 targets, but their roles as HIV-1 reservoirs remain controversial. Here we provide additional evidence for the susceptibility of several distinct HSPC subpopulations to HIV-1 infection in vitro and in vivo. In vitro infection experiments of HSPCs were performed with different HIV-1 Env-pseudotyped lentiviral particles and with replication-competent HIV-1. Low-level infection/transduction of HSPCs, including hematopoietic stem cells (HSCs) and multipotent progenitors (MPP), was observed, preferentially via CXCR4, but also via CCR5-mediated entry. Multi-lineage colony formation in methylcellulose assays and repetitive replating of transduced cells provided functional proof of susceptibility of primitive HSPCs to HIV-1 infection. Further, the access to bone marrow samples from HIV-positive individuals facilitated the detection of HIV-1 gag cDNA copies in CD34+ cells from eight (out of eleven) individuals, with at least six of them infected with CCR5-tropic HIV-1 strains. In summary, our data confirm that primitive HSPC subpopulations are susceptible to CXCR4- and CCR5-mediated HIV-1 infection in vitro and in vivo, which qualifies these cells to contribute to the HIV-1 reservoir in patients.
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Storms, Robert W., Patrick D. Green, Kristine M. Safford, Donna Niedzwiecki, Christopher R. Cogle, O. Michael Colvin, Nelson J. Chao, Henry E. Rice, and Clayton A. Smith. "Distinct hematopoietic progenitor compartments are delineated by the expression of aldehyde dehydrogenase and CD34." Blood 106, no. 1 (July 1, 2005): 95–102. http://dx.doi.org/10.1182/blood-2004-09-3652.
Full textAbstract:
A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSClo) and high expression of aldehyde dehydrogenase (ALDHbr). Many SSClo ALDHbr cells coexpress CD34; however, other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets, purified ALDHbr CD34+, ALDHneg CD34+, and ALDHbr CD34neg UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDHbr CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison, ALDHneg CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDHbr CD34neg cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development.
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Wang, Jean C. Y., Monica Doedens, and John E. Dick. "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 89, no. 11 (June 1, 1997): 3919–24. http://dx.doi.org/10.1182/blood.v89.11.3919.
Full textAbstract:
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.
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Chelucci, C., HJ Hassan, C. Locardi, D. Bulgarini, E. Pelosi, G. Mariani, U. Testa, M. Federico, M. Valtieri, and C. Peschle. "In vitro human immunodeficiency virus-1 infection of purified hematopoietic progenitors in single-cell culture." Blood 85, no. 5 (March 1, 1995): 1181–87. http://dx.doi.org/10.1182/blood.v85.5.1181.bloodjournal8551181.
Full textAbstract:
Uni- or multi-lineage suppression of hematopoiesis is observed in the majority of acquired immunodeficiency syndrome (AIDS) patients. The mechanism(s) underlying these abnormalities is not understood: particularly, the human immunodeficiency virus (HIV) infection of hematopoietic progenitor and stem cells (HPCs/HSCs) is highly controversial. We report that CD34+ HPCs from adult peripheral blood (PB) are in part CD4+ and susceptible to in vitro HIV infection. Primitive CD34+ HPCs were approximately 80% purified from PB. Double labeling for CD34 and CD4 membrane antigens was shown for 5% to 20% of the purified cells, thus suggesting their potential susceptibility to HIV-1 infection. The enriched HPC population, challenged with purified or unpurified HIV-1 strains, was cloned in unicellular methylcellulose culture. The single colonies generated by erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), and granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-GEMM) were analyzed for the presence of HIV, ie, for gag DNA, tat mRNA, and p24 protein by PCR, reverse transcription PCR (RT-PCR), and enzyme-linked immunosorbent assay, respectively. In the first series of experiments incubation of HPCs with HIV-1 at multiplicities of infection (MOI) ranging from 0.01 to 10 TCID50/cell consistently yielded an 11% to 17% infection efficiency of BFU-E-generated colonies, thus indicating the sensitivity of HPCs to in vitro HIV infection. An extensive series of experiments was then performed on HPCs challenged with HIV at 0.1 MOI level. In the initial studies proviral gag sequences were detected in 9.2% of 121 analyzed CFU-GM colonies. In further experiments tat mRNA was monitored in 17% and 23% of BFU-E and CFU-GM colonies, respectively, but never in CFU-GEMM clones. Finally, 12% of CFU-GM clones and rare erythroid bursts were shown to be positive for the p24 viral protein. In control studies, purified HPCs grown in liquid suspension culture were induced to terminal unilineage erythroid, monocytic, or granulocytic differentiation: monocytes were consistently HIV-infected, whereas mature-terminal erythroblasts and granulocytes were not. Our observations indicate that a minority of primitive HPCs, but not of the multipotent type, is susceptible to in vitro HIV infection. These observations may reflect on the in vivo hematopoietic impairment in AIDS patients; more important, they provide an experimental model for studies on HIV hematopoietic infection and in vitro tests for anti-HIV HSC gene therapy.
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Sirven, Aude, Françoise Pflumio, Véronique Zennou, Monique Titeux, William Vainchenker, Laure Coulombel, Anne Dubart-Kupperschmitt, and Pierre Charneau. "The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells." Blood 96, no. 13 (December 15, 2000): 4103–10. http://dx.doi.org/10.1182/blood.v96.13.4103.
Full textAbstract:
Abstract Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34+ hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).
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Sirven, Aude, Françoise Pflumio, Véronique Zennou, Monique Titeux, William Vainchenker, Laure Coulombel, Anne Dubart-Kupperschmitt, and Pierre Charneau. "The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells." Blood 96, no. 13 (December 15, 2000): 4103–10. http://dx.doi.org/10.1182/blood.v96.13.4103.h8004103_4103_4110.
Full textAbstract:
Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34+ hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).
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Cobaleda, C., N. Gutiérrez-Cianca, J. Pérez-Losada, T. Flores, R. Garcı́a-Sanz, M. González, and I. Sánchez-Garcı́a. "A primitive hematopoietic cell is the target for the leukemic transformation in human Philadelphia-positive acute lymphoblastic leukemia." Blood 95, no. 3 (February 1, 2000): 1007–13. http://dx.doi.org/10.1182/blood.v95.3.1007.003k35_1007_1013.
Full textAbstract:
BCR-ABL is a chimeric oncogene generated by translocation of sequences from the chromosomal counterpart (c-ABLgene) on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, BCR-ABLp190 and BCR-ABLp210, are produced that are characteristic of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph1-ALL). In CML, the transformation occurs at the level of pluripotent stem cells. However, Ph1-ALL is thought to affect progenitor cells with lymphoid differentiation. Here we demonstrate that the cell capable of initiating human Ph1-ALL in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID), termed SCID leukemia–initiating cell (SL-IC), possesses the differentiative and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell. The SL-ICs from all Ph1-ALL analyzed, regardless of the heterogeneity in maturation characteristics of the leukemic blasts, were exclusively CD34+CD38−, which is similar to the cell-surface phenotype of normal SCID-repopulating cells. This indicates that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation in Ph1-ALL.
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Terpstra, Wim, Henk Rozemuller, Dimitri A. Breems, Elwin J. C. Rombouts, Arie Prins, David J. P. FitzGerald, Robert J. Kreitman, et al. "Diphtheria Toxin Fused to Granulocyte-Macrophage Colony-Stimulating Factor Eliminates Acute Myeloid Leukemia Cells With the Potential to Initiate Leukemia in Immunodeficient Mice, But Spares Normal Hemopoietic Stem Cells." Blood 90, no. 9 (November 1, 1997): 3735–42. http://dx.doi.org/10.1182/blood.v90.9.3735.
Full textAbstract:
Abstract We studied the cell kill induced by granulocyte-macrophage colony-stimulating factor (GM-CSF ) fused to Diphtheria Toxin (DT-GM-CSF ) in acute myeloid leukemia (AML) samples and in populations of normal primitive hemopoietic progenitor cells. AML samples from three patients were incubated in vitro with 100 ng/mL DT-GM-CSF for 48 hours, and AML cell kill was determined in a proliferation assay, a clonogenic assay colony-forming unit-AML (CFU-AML) and a quantitative long-term bone marrow (BM) culture ie, the leukemic-cobblestone area forming cell assay (L-CAFC). To measure an effect on cells with in vivo leukemia initiating potential DT-GM-CSF exposed AML cells were transplanted into immunodeficient mice. In two out of three samples it was shown that all AML subsets, including those with long-term abilities in vivo (severe combined immunodeficient mice) and in vitro (L-CAFC assay) were reduced in number by DT-GM-CSF. Cell kill induced by DT-GM-CSF could be prevented by coincubation with an excess of GM-CSF, demonstrating that sensitivity to DT-GM-CSF is specifically mediated by the GM-CSF receptor. Therefore, binding and internalization of GM-CSF probably occur in immature AML precursors of these two cases of AML. The third AML sample was not responsive to either GM-CSF or DT-GM-CSF. The number of committed progenitors of normal bone marrow (burst-forming unit-erythroid, colony-forming unit granulocyte- macrophage, and cobble stone area forming cell [CAFC] week 2) and also the number of cells with long-term repopulating ability, assayed as week 6 CAFC, were unchanged after exposure to DT-GM-CSF (100 ng/mL, 48 hours). These studies show that DT-GM-CSF may be used to eliminate myeloid leukemic cells with long-term potential in vitro and in immunodeficient mice, whereas normal hemopoietic stem cells are spared.
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Davis, Brian M., Laurent Humeau, Vladimir Slepushkin, Gwendolyn Binder, Lauren Korshalla, Yajin Ni, E. Oluwakemi Ogunjimi, Lan-Fei Chang, Xiaobin Lu, and Boro Dropulic. "ABC transporter inhibitors that are substrates enhance lentiviral vector transduction into primitive hematopoietic progenitor cells." Blood 104, no. 2 (July 15, 2004): 364–73. http://dx.doi.org/10.1182/blood-2003-07-2363.
Full textAbstract:
Abstract High gene transfer efficiencies have been difficult to achieve in hematopoietic progenitor cells (HPCs) but are important to therapeutic success of HPC gene therapy. Efficient gene transfer is especially challenging with use of column-purified vector for clinical application, as opposed to centrifuged vector commonly used for research. We investigated novel approaches to increase transduction by using a clinically applicable protocol and quantities of column-purified lentiviral vector. Recognizing the association of adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporters with HPC biology, we investigated the effect of transporter inhibitors on transduction. We found the ABC transporter inhibitor verapamil improved transduction efficiency 2- to 6-fold into CD34+ cells isolated from mobilized peripheral blood, bone marrow, and cord blood. Verapamil also improved transduction in human SCID (severe combined immunodeficient) repopulating cell (SRC) transduction 3- to 4-fold, resulting in 80% to 90% transduction levels in mice receiving primary and secondary transplants without alterations in multilineage reconstitution. Additional ABC transporter substrate inhibitors like quinidine, diltiazem, and ritonavir also enhanced transduction 2- to 3-fold, although ABC transporter inhibitors that are not substrates did not. Enhanced transduction was not observed in mature hematopoietic cells, neurospheres, mesenchymal stem cells, or hepatocytes. Enhancement of transduction in HPCs was observed with vesicular stomatitis virus-G (VSV-G)-pseudotyped lentiviral vector but not with vector pseudotyped with RD114. Thus, we present a new approach for efficient delivery to primitive HPCs by VSV-G-pseudotyped lentiviral vectors. (Blood. 2004;104:364-373)
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Wong, Wan Man, Ingbritt Åstrand-Grundström, Hong Qian, and Marja Ekblom. "Differential Expression of Alpha2 Integrin In Human Bone Marrow and Cord Blood CD34+CD38- Progenitors and Stem Cells Reconstituting NOD/SCID-IL2Rgammacnull Mice." Blood 116, no. 21 (November 19, 2010): 2609. http://dx.doi.org/10.1182/blood.v116.21.2609.2609.
Full textAbstract:
Abstract Abstract 2609 Human primitive stem cells reside in the CD34+CD38- fraction in cord blood and bone marrow. However, there is a high level of heterogeneity in these cell fractions, and the phenotype of the rare primitive stem cells remains poorly defined. We have studied expression of integrin alpha2 chain, a member of a family of beta1 integrin cell adhesion receptors, in CD34+CD38- cells in adult bone marrow and cord blood. >90% of bone marrow CD34+CD38- cells and >95% of CD34+CD38-CD90+ cells, enriched in long-term in vivo reconstituting stem cells (Majeti et al., Cell Stem Cell 1:635, 2007) expressed the integrin alpha2 chain. In contrast, in cord blood CD34+CD38- and the CD34+CD38-CD90+ subpopulation, the integrin alpha2 chain was expressed only in 37.1+/− 5.3% and 32.2+/− 4.9% of the cells (mean+/− SD), respectively. To determine whether integrin alpha2 expression could be used to identify functionally distinct stem and progenitor cell populations in cord blood and bone marrow, we isolated CD34+CD38- integrin alpha2+ and alpha2- cells by flow cytometry and analyzed these by in vivo transplantation into immunodeficient NOD/SCID-IL2Rgammacnull (NSG) mice and by in vitro progenitor cell assays (long-term culture initiating cell, LTC-IC, and colony assays). Transplantation of cord blood CD34+CD38- integrin alpha2+ cells resulted in significantly higher level of human CD45+ (p<0.05), myeloid (p<0.01) and CD34+ (p<0.05) cell engraftment at 16–18 weeks after transplantation than integrin alpha2- cells (reconstitution/300 cells in age and sex matched recipients, Figure 1). In contrast, there were no differences in reconstitution at 12 weeks in mice transplanted with cord blood CD34+CD38- alpha2+ and alpha2- cells. Because of lower engraftment capacity of adult bone marrow cells in immunodeficient mice, bone marrow CD34+CD38- cells were analyzed after intra-bone transplantation. After 12 weeks only few mice transplanted with the CD34+CD38- integrin alpha2+ cells but none transplanted with the corresponding alpha2- cells were reconstituted with human CD45+ or myeloid cells at a level '0.1%. The LTC-IC progenitors within the CD34+CD38- populations, assayed after 6 weeks culture on stroma, in adult bone marrow were highly enriched in the alpha2+ as compared with alpha2- fraction (mean +/− SD 96.7+/− 57.0 and 0.2+/− 0.6 colonies/50 cells, respectively), whereas in cord blood they resided equally in both integrin alpha2+ and alpha2- cell fractions (mean +/− SD 77.4+/− 59.4 and 108.8+/− 96.0 colonies/50 cells, respectively). The lineage committed CFU-GM and BFU-E progenitors in adult bone marrow were within integrin CD34+CD38- alpha2+ and alpha2- fractions (mean +/− SD 8.5+/− 5.3 and 14.9+/− 16.2 colonies/100 cells, respectively), whereas in cord blood they were significantly enriched in the alpha2- fraction (mean +/− SD 9.3+/− 5.5 and 22.0+/− 6.7 colonies/100 cells in alpha2+ and alpha2- fractions, respectively). Taken together, our results show expression of integrin alpha2 receptor in most of the primitive cord blood long-term in vivo reconstituting stem cells, with a gradual loss of the integrin alpha2 receptor during maturation to short term in vivo reconstituting stem cells, LTC-IC and lineage committed progenitors. Furthermore, our findings show distinct ontogeny-related differences in the expression of the integrin alpha2 receptor in the functionally defined primitive and lineage-committed CD34+CD38- progenitors, indicating differences in cellular interactions of cord blood and bone marrow progenitors with the hematopoietic niches. Disclosures: Ekblom: Bristol-Myers Squibb: Honoraria.
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Holyoake, Tessa, Xiaoyan Jiang, Connie Eaves, and Allen Eaves. "Isolation of a Highly Quiescent Subpopulation of Primitive Leukemic Cells in Chronic Myeloid Leukemia." Blood 94, no. 6 (September 15, 1999): 2056–64. http://dx.doi.org/10.1182/blood.v94.6.2056.
Full textAbstract:
Abstract Chronic myeloid leukemia (CML) is characterized by an increased proliferative activity of the leukemic progenitors that produce an elevated number of mature granulocytes. Nevertheless, cell cycle-active agents, even in very high doses, are alone unable to eradicate the leukemic clone, suggesting the presence of a rare subset of quiescent leukemic stem cells. To isolate such cells, we first used Hoechst 33342 and Pyronin Y staining to obtain viable G0 and G1/S/G2/M fractions of CD34+cells by fluorescence-activated cell sorting (FACS) from 6 chronic-phase CML patients’ samples and confirmed the quiescent and cycling status of the 2 fractions by demonstration of expected patterns of Ki-67 and D cyclin expression. Leukemic (Ph+/BCR-ABL+) cells with in vitro progenitor activity and capable of engrafting immunodeficient mice were identified in the directly isolated G0 cells. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that many leukemic CD34+ G0cells also expressed BCR-ABL mRNA. CD34+ from 8 CML patients were also labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) before being cultured (with and without added growth factors) to allow viable cells that had remained quiescent (ie, CFSE+) after 4 days to be retrieved by FACS. Leukemic progenitors were again detected in all quiescent populations isolated by this second strategy, including those exposed to a combination of flt3-ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These findings provide the first direct and definitive evidence of a deeply but reversibly quiescent subpopulation of leukemic cells in patients with CML with both in vitro and in vivo stem cell properties.