The Experts below are selected from a list of 1824189 Experts worldwide ranked by ideXlab platform
Kazunari Kaneko - One of the best experts on this subject based on the ideXlab platform.
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cd133 is a positive marker for a distinct class of primitive human cord blood derived cd34 negative hematopoietic stem cells
Leukemia, 2014Co-Authors: Masaya Takahashi, Yoshikazu Matsuoka, Keisuke Sumide, Ryusuke Nakatsuka, Tatsuya Fujioka, Hirao Kohno, Yutaka Sasaki, K Matsui, Hiroaki Asano, Kazunari KanekoAbstract:The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34+CD38− SRCs. However, the purification level of rare CD34− SRCs in 18 lineage marker-negative (Lin−) CD34− cells (1/1000) is still very low compared with that of CD34+CD38− SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34− SRCs. Using 18Lin−CD34− cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34− SRCs and succeeded in highly purifying primitive human CD34− HSCs. The limiting dilution analysis demonstrated that the incidence of CD34− SRCs in 18Lin−CD34−CD133+ cells was 1/142, which is the highest level of purification of these unique CD34− HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin−CD34− cells, as well as 18Lin−CD34+ cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34+ and CD34− SRCs.
Dennis E. Van Epps - One of the best experts on this subject based on the ideXlab platform.
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phenotypic analysis and characterization of cd34 cells from normal human bone marrow cord blood peripheral blood and mobilized peripheral blood from patients undergoing autologous stem cell transplantation
Clinical Immunology and Immunopathology, 1994Co-Authors: James G. Bender, S F Williams, Kristen L Unverzagt, D E Walker, Stephen D Smith, Dennis E. Van EppsAbstract:Abstract Single- and multicolor flow cytometry were used to define progenitor subsets in normal human bone marrow and peripheral blood, cord blood, and blood following mobilization of CD34+ progenitor cells by cyclophosphamide or cyclophosphamide/etoposide/G-CSF treatment. CD34 cells were quantitated and subsets of CD34+ cells were defined by coexpression of CD33, CD13, CD10, CD19, CD45RA, and CD71. Myeloid and erythroid progenitors were quantitated by sorting single CD34+ cells into individual wells of 96-well plates containing methylcellulose, IL-3, GM-CSF, G-CSF, IL-6, and erythropoietin. Comparative studies of CD34 cells showed that the percentage of CD34+ mononuclear cells was greatest in blood samples from patients following mobilization treatment with cyclophosphamide/etoposide/G-CSF averaging 2%. By comparison, the remaining sample groups ranged from 1.68 to 0.15% CD34 cells in this order, bone marrow > cord blood > cyclophosphamide mobilized blood > peripheral blood. Comparison of CD34 cells per milliliter of bone marrow or blood showed a range of 22.4 × 104 to 0.65 × 104/ml in the following order, bone marrow > chemotherapy/etoposide/G-CSF > cord blood > cyclophosphamidemobilized blood. Comparative analysis of CD34 subsets from different sources showed significant differences, particularly bone marrow and blood samples. A distinct population of CD34+ CD19+ (Leu 12) CD10+ (CALLA) pre-B lymphocyte cells was defined in bone marrow with lower side and forward light scatter characteristics and was variable between donors (29.8 ± 16.9%, mean ± 1 SD; range, 3-54%; n = 8). This population was not found to a significant degree in blood and also expressed CD45RA (Leu 18). Coexpression studies of CD45RA and CD71 (transferrin receptor) expression on CD34+ cells defined a CD45RA-CD71+ population containing 89 ± 6.3% (n = 4) BFU-E and a CD45RA+CD71+ population that contained all CFU-GM (n = 4). LeuM7 (CD13) stained a larger percentage to a greater intensity than MY7 (CD13). Coexpression of CD45RA (Leu 18) and CD13 (LeuM7) defined a subset of CD13+ CD45RA+ cells enriched for CFU-GM and CFU-M with a cloning efficiency of 31%. Coexpression of CD33 (MY9) and CD13 (MY7) defined a population that was predominantly CFU-GM with a cloning efficiency of 38%. These studies define CD34+ phenotypes containing pure populations of B lymphocyte, granulocyte-macrophage, or erythroid progenitors and demonstrate the utility of multiparameter flow cytometry to define lineage-committed CD34+ cells.
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characterization of chemotherapy mobilized peripheral blood progenitor cells for use in autologous stem cell transplantation
Bone Marrow Transplantation, 1992Co-Authors: James G. Bender, S F Williams, Sage R Myers, D Nottleman, Kristen L Unverzagt, D E Walker, L B To, Dennis E. Van EppsAbstract:: Twenty patients were treated with chemotherapy to mobilize progenitors into the blood. Peripheral blood stem cells were quantitated in peripheral blood or leukapheresis products using colony assays and flow cytometric measurement of CD34+ cells. In four patients where complete sets of serial samples were obtained, the appearance of CD34+ cells preceded the increase in CFU-GM by 24-48 h. Peak levels of CD34+ cells ranged from 0.6-5% and coincided with the peak increase in CFU-GM. Mobilized CD34+ cells contained subsets expressing CD33, CD13, CD45RA, CD38, HLA-DR, CD61 and CD41. Subsets of CD34+ cells expressing CD33, CD13, or CD45RA represent committed myeloid progenitors. In contrast to bone marrow CD34+ cells, few mobilized CD34+ cells expressed CD71, CD7, CD19 or CD10. Prompt engraftment of granulocytes greater than 500 x 10(6)/l at a median of 13 days and platelets greater than 50 x 10(9)/l at a median of 15 days was observed in patients reconstituted with mobilized cells. These data indicate that CD34+ cells mobilized during recovery from chemotherapy are predominantly myeloid in phenotype and contain few actively proliferating cells or cells with lymphoid phenotypes.
Masaya Takahashi - One of the best experts on this subject based on the ideXlab platform.
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cd133 is a positive marker for a distinct class of primitive human cord blood derived cd34 negative hematopoietic stem cells
Leukemia, 2014Co-Authors: Masaya Takahashi, Yoshikazu Matsuoka, Keisuke Sumide, Ryusuke Nakatsuka, Tatsuya Fujioka, Hirao Kohno, Yutaka Sasaki, K Matsui, Hiroaki Asano, Kazunari KanekoAbstract:The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34+CD38− SRCs. However, the purification level of rare CD34− SRCs in 18 lineage marker-negative (Lin−) CD34− cells (1/1000) is still very low compared with that of CD34+CD38− SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34− SRCs. Using 18Lin−CD34− cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34− SRCs and succeeded in highly purifying primitive human CD34− HSCs. The limiting dilution analysis demonstrated that the incidence of CD34− SRCs in 18Lin−CD34−CD133+ cells was 1/142, which is the highest level of purification of these unique CD34− HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin−CD34− cells, as well as 18Lin−CD34+ cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34+ and CD34− SRCs.
Keisuke Sumide - One of the best experts on this subject based on the ideXlab platform.
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human cord blood derived cd34 negative hematopoietic stem cells hscs a new class of hscs in the human hsc hierarchy
Blood, 2015Co-Authors: Keisuke Sumide, Yoshikazu Matsuoka, Ryusuke Nakatsuka, Tatsuya Fujioka, Yutaka Sasaki, Hiroaki Asano, Hiroshi Kawamura, Yoshiaki SonodaAbstract:Background . We previously identified very primitive human cord blood (CB)-derived CD34-negative (CD34 - ) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). A series of our studies suggests that the CD34 - SRCs that we identified are a distinct class of primitive hematopoietic stem cells (HSCs), which show myeloid-biased long-term repopulating capacity, suggesting that they are at the apex of the human HSC hierarchy (Blood Cancer J 2015:5,e290). We recently developed a high-resolution purification method for primitive CD34 - SRCs using 18 lineage (Lin)-specific antibodies, which can enrich CD34 - SRC at the 1/1,000 level (Exp Hematol 2011:39;203). We then identified CD133 as a positive marker of human CB-derived CD34 +/- SRCs. Moreover, limiting dilution analyses (LDAs) demonstrated that the frequencies of SRCs in the 18Lin - CD34 +/- CD133 + fractions were 1/100 and 1/140, respectively (Leukemia 2014:28;1308). Since we aim to purify the CD34 - SRCs (HSCs) at the single cell level, it was necessary to identify other specific positive markers for CD34 - SRCs. We extensively analyzed the expression of candidate positive markers in the 18Lin - CD34 - cell population by FACS. Finally, we identified glycosylphosphatidyl-inoshitol-anchored surface protein (GPI-80) as a useful marker of human CB-derived CD34 +/- SRCs and succeeded in highly purifying primitive human CD34 +/- SRCs to the level of 1/20 cells. Aim . We herein attempted to purify CD34 +/- SRCs to the single cell level in order to precisely characterize the CD34 - SRCs (HSCs) in the human HSC hierarchy. Materials and Methods . We first developed an ultra-high resolution purification method using two reliable markers for CD34 +/- SRCs, including CD133 and GPI-80. Namely, we sorted 18Lin - CD34 + CD38 - CD133 + GPI-80 + (34 + 38 - 133 + 80 + )and 18Lin - CD34 - CD133 + GPI-80 + (34 - 133 + 80 + )cells by FACS. Thereafter, these two fractions of cells were transplanted by the IBMI technique into NOD/Shi-scid/IL-2Rγc null (NOG) mice to investigate their long-term repopulating capacities. We ultimately performed single cell transplantations and analyzed their human hematopoietic cell reconstitution for up to 20 weeks. Finally, we analyzed their gene expression profiles, including the key genes for the self-renewal and maintenance of HSCs of single 34 + 38 - 133 + 80 + and 34 - 133 + 80 + cells using a BioMark System (Fluidigm). Results . Approximately 15% of the 34 + 38 - 133 + and 34 - 133 + cells expressed GPI-80. These highly purified cells showed very immature blast-like morphologies. These two fractions of cells were then transplanted into NOG mice by IBMI. We performed primary and secondary transplantations for up to 40 weeks. In the results, all of the mice received 200 34 + 38 - 133 + 80 + (n=25)and 34 - 133 + 80 + (n=23) cells were repopulated with human CD45 + cells (Mean % of human CD45 + cells, 47% vs. 35%), including CD34 + , CD19 + and CD33 + cells. Most of the secondary transplanted mice were also repopulated with human CD45 + cells with multi-lineage reconstitution (Mean % of human CD45 + cells, 0.4% vs. 10%). An LDA is currently underway; however, the frequencies of CD34 +/- SRCs in the 34 + 38 - 133 + 80 + and 34 - 133 + 80 + cells are estimated to be 1/5-1/10. Interestingly, significant numbers of the recipient mice that received single cells displayed multi-lineage human cell repopulation at 20 weeks after transplantation. In order to provide an independent line of evidence for characterizing our highly purified CD34 +/- SRCs, we analyzed the gene expression profiles of these two types of SRCs at the single cell level. The principle component analysis clearly demonstrated that the gene expression profiles of individual CD34 + and CD34 - SRCs were clearly different. Both SRCs expressed high levels of HSC maintenance genes, including RUNX1, TAL1, BMI1 and MYBC. Very interestingly, CD34 + SRCs expressed a high level of ETV6. In contrast, CD34 - SRCs expressed higher levels of EZH2 and RING1. These results suggest that different mechanisms control HSC self-renewal and maintenance, as well as epigenetic regulation in these two SRCs. Conclusion . Wedeveloped an ultra-high resolution purification method using two markers for CD34 +/- SRCs, including CD133 and GPI-80. The precise single cell-based analysis allows us to map CD34 - SRCs (HSCs) at the apex of the human HSC hierarchy. Disclosures No relevant conflicts of interest to declare.
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cd133 is a positive marker for a distinct class of primitive human cord blood derived cd34 negative hematopoietic stem cells
Leukemia, 2014Co-Authors: Masaya Takahashi, Yoshikazu Matsuoka, Keisuke Sumide, Ryusuke Nakatsuka, Tatsuya Fujioka, Hirao Kohno, Yutaka Sasaki, K Matsui, Hiroaki Asano, Kazunari KanekoAbstract:The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34+CD38− SRCs. However, the purification level of rare CD34− SRCs in 18 lineage marker-negative (Lin−) CD34− cells (1/1000) is still very low compared with that of CD34+CD38− SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34− SRCs. Using 18Lin−CD34− cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34− SRCs and succeeded in highly purifying primitive human CD34− HSCs. The limiting dilution analysis demonstrated that the incidence of CD34− SRCs in 18Lin−CD34−CD133+ cells was 1/142, which is the highest level of purification of these unique CD34− HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin−CD34− cells, as well as 18Lin−CD34+ cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34+ and CD34− SRCs.
Yutaka Sasaki - One of the best experts on this subject based on the ideXlab platform.
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human cord blood derived cd34 negative hematopoietic stem cells hscs a new class of hscs in the human hsc hierarchy
Blood, 2015Co-Authors: Keisuke Sumide, Yoshikazu Matsuoka, Ryusuke Nakatsuka, Tatsuya Fujioka, Yutaka Sasaki, Hiroaki Asano, Hiroshi Kawamura, Yoshiaki SonodaAbstract:Background . We previously identified very primitive human cord blood (CB)-derived CD34-negative (CD34 - ) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). A series of our studies suggests that the CD34 - SRCs that we identified are a distinct class of primitive hematopoietic stem cells (HSCs), which show myeloid-biased long-term repopulating capacity, suggesting that they are at the apex of the human HSC hierarchy (Blood Cancer J 2015:5,e290). We recently developed a high-resolution purification method for primitive CD34 - SRCs using 18 lineage (Lin)-specific antibodies, which can enrich CD34 - SRC at the 1/1,000 level (Exp Hematol 2011:39;203). We then identified CD133 as a positive marker of human CB-derived CD34 +/- SRCs. Moreover, limiting dilution analyses (LDAs) demonstrated that the frequencies of SRCs in the 18Lin - CD34 +/- CD133 + fractions were 1/100 and 1/140, respectively (Leukemia 2014:28;1308). Since we aim to purify the CD34 - SRCs (HSCs) at the single cell level, it was necessary to identify other specific positive markers for CD34 - SRCs. We extensively analyzed the expression of candidate positive markers in the 18Lin - CD34 - cell population by FACS. Finally, we identified glycosylphosphatidyl-inoshitol-anchored surface protein (GPI-80) as a useful marker of human CB-derived CD34 +/- SRCs and succeeded in highly purifying primitive human CD34 +/- SRCs to the level of 1/20 cells. Aim . We herein attempted to purify CD34 +/- SRCs to the single cell level in order to precisely characterize the CD34 - SRCs (HSCs) in the human HSC hierarchy. Materials and Methods . We first developed an ultra-high resolution purification method using two reliable markers for CD34 +/- SRCs, including CD133 and GPI-80. Namely, we sorted 18Lin - CD34 + CD38 - CD133 + GPI-80 + (34 + 38 - 133 + 80 + )and 18Lin - CD34 - CD133 + GPI-80 + (34 - 133 + 80 + )cells by FACS. Thereafter, these two fractions of cells were transplanted by the IBMI technique into NOD/Shi-scid/IL-2Rγc null (NOG) mice to investigate their long-term repopulating capacities. We ultimately performed single cell transplantations and analyzed their human hematopoietic cell reconstitution for up to 20 weeks. Finally, we analyzed their gene expression profiles, including the key genes for the self-renewal and maintenance of HSCs of single 34 + 38 - 133 + 80 + and 34 - 133 + 80 + cells using a BioMark System (Fluidigm). Results . Approximately 15% of the 34 + 38 - 133 + and 34 - 133 + cells expressed GPI-80. These highly purified cells showed very immature blast-like morphologies. These two fractions of cells were then transplanted into NOG mice by IBMI. We performed primary and secondary transplantations for up to 40 weeks. In the results, all of the mice received 200 34 + 38 - 133 + 80 + (n=25)and 34 - 133 + 80 + (n=23) cells were repopulated with human CD45 + cells (Mean % of human CD45 + cells, 47% vs. 35%), including CD34 + , CD19 + and CD33 + cells. Most of the secondary transplanted mice were also repopulated with human CD45 + cells with multi-lineage reconstitution (Mean % of human CD45 + cells, 0.4% vs. 10%). An LDA is currently underway; however, the frequencies of CD34 +/- SRCs in the 34 + 38 - 133 + 80 + and 34 - 133 + 80 + cells are estimated to be 1/5-1/10. Interestingly, significant numbers of the recipient mice that received single cells displayed multi-lineage human cell repopulation at 20 weeks after transplantation. In order to provide an independent line of evidence for characterizing our highly purified CD34 +/- SRCs, we analyzed the gene expression profiles of these two types of SRCs at the single cell level. The principle component analysis clearly demonstrated that the gene expression profiles of individual CD34 + and CD34 - SRCs were clearly different. Both SRCs expressed high levels of HSC maintenance genes, including RUNX1, TAL1, BMI1 and MYBC. Very interestingly, CD34 + SRCs expressed a high level of ETV6. In contrast, CD34 - SRCs expressed higher levels of EZH2 and RING1. These results suggest that different mechanisms control HSC self-renewal and maintenance, as well as epigenetic regulation in these two SRCs. Conclusion . Wedeveloped an ultra-high resolution purification method using two markers for CD34 +/- SRCs, including CD133 and GPI-80. The precise single cell-based analysis allows us to map CD34 - SRCs (HSCs) at the apex of the human HSC hierarchy. Disclosures No relevant conflicts of interest to declare.
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cd133 is a positive marker for a distinct class of primitive human cord blood derived cd34 negative hematopoietic stem cells
Leukemia, 2014Co-Authors: Masaya Takahashi, Yoshikazu Matsuoka, Keisuke Sumide, Ryusuke Nakatsuka, Tatsuya Fujioka, Hirao Kohno, Yutaka Sasaki, K Matsui, Hiroaki Asano, Kazunari KanekoAbstract:The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34+CD38− SRCs. However, the purification level of rare CD34− SRCs in 18 lineage marker-negative (Lin−) CD34− cells (1/1000) is still very low compared with that of CD34+CD38− SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34− SRCs. Using 18Lin−CD34− cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34− SRCs and succeeded in highly purifying primitive human CD34− HSCs. The limiting dilution analysis demonstrated that the incidence of CD34− SRCs in 18Lin−CD34−CD133+ cells was 1/142, which is the highest level of purification of these unique CD34− HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin−CD34− cells, as well as 18Lin−CD34+ cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34+ and CD34− SRCs.