Direct Isolation and Expansion of Human CD34<sup>+</sup>Hematopoietic Stem Cells

Lebkowski, Jane; Schain, Lisa R.; Harvey, Mark J.; Moseley, Annemarie; Okarma, Thomas B.

doi:10.1089/scd.1.1993.2.339

Cited by 9 publications

(5 citation statements)

References 4 publications

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“…Positive selection of CD34 expressing cells has been applied to reduce tumour cell contamination in both bone marrow and peripheral blood autografts [16, 17]. Several methods for CD34 selection are available, such as: immunomagnetic beads directly coated with the anti‐CD34 MoAb BI‐3C5 (class I: Dynal, Oslo, Norway) [9]; beads coated with anti‐mouse antibodies that recognize 9C5 (class II) bound cells (Isolex: Baxter, Irvine, CA, USA) [18]; Sepharose particles with 12.8 (class I) for column immunoabsorption (Cellpro Inc., Bothell, WA, USA) [19]; magnetic microspheres with QBEND‐10 (class II, MACS: Miltenyi Biotec Gmbh, Bergisch Gladbach, Germany) [20]; and panning on culture flasks using ICH3 (class II: Applied Immune Systems, Menlo Park, CA, USA) [21]. According to the literature CD34 selection is hampered by low yields, 35–70% for the laboratory scale methods and 45–50% for the grafting materials [22].…”

Section: Introductionmentioning

confidence: 99%

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34⁺ Bone Marrow and Peripheral Blood Stem Cells

et al. 1998

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Positive selection of CD34þ cells is an attractive approach to reduce tumour cell contamination in bone marrow (BM) and peripheral blood progenitor cell (PBPC) autografts in malignancies not expressing CD34. All current selection methods use monoclonal antibodies (MoAbs) specific for the class I or class II CD34 epitopes, while for detection most investigators use class III MoAbs. Since the distribution of the different CD34 epitopes on haematopoietic progenitors differs, we studied their significance in CD34 þ selection procedures. Testing MoAbs against class I, II and III CD34 epitopes on normal BM we observed that Ϯ 23% of class III positive cells was class I negative. A higher expression of the class III epitope compared with classes I or II was observed on the KG1 cell line, whereas no differences in binding capability were found. The class III epitope anti-CD34, 561, was compared with the class I epitope anti-CD34, BI-3C5, both coupled to M450 Dynabeads. The yield of CD34 þ cells obtained with the 561 beads was 1.7% of the mononuclear cells versus 0.95% using the class I epitope, a 1.95-fold increase (1.3-2.7), whereas the purity was similar (96% in both cases). The absolute number of CD34 þ cells was therefore twofold higher after 561 selection, including cells with a more mature phenotype. In single cell assay comparable numbers of highly proliferative progenitors but higher numbers of differentiated colonies per phenotypic subfraction were measured. In conclusion, M450 beads coated with the 561 anti-class III CD34 epitope are more efficient in isolating CD34 þ cells from bone marrow, probably due to a broader distribution of the class III epitope.

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Section: Introductionmentioning

confidence: 99%

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34⁺ Bone Marrow and Peripheral Blood Stem Cells

et al. 1998

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“…Recently, the possibility of culturing hematopoietic cells in serum‐free medium [22] has provided biochemically defined culture conditions. Several formulae for serum‐depleted media supporting clonogenic growth in semisolid media [23, 24], liquid cultures of bone marrow [25] or expansion of CD34 + cells in liquid culture [26] have been published. In addition, based on various techniques using monoclonal anti‐CD34 antibodies, the CD34 + progenitor cells collected by leukapheresis can be highly enriched to a purity of more than 95%, thus providing ideal targets to assess the effects of cytokines in early hematopoiesis.…”

Section: Introductionmentioning

confidence: 99%

Effects of Recombinant Human Thrombopoietin Alone and in Combination with Erythropoietin and Early‐Acting Cytokines on Human Mobilized Purified CD34 ⁺ Progenitor Cells Cultured in Serum‐Depleted Medium

et al. 1997

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The effects of recombinant thrombopoietin (TPO) alone and in combination with erythropoietin (EPO) and early-acting cytokines such as interleukin 3 (IL-3), stem cell factor (SCF) and GM-CSF on highly purified mobilized human CD34 + progenitor cells were studied in a serum-depleted culture system. Eight leukapheresis samples were cultured for seven days and analyzed; aliquots were replated and re-evaluated on day 12. Three-color flow cytometry was used together with morphologic analysis to determine proliferation and megakaryocytic or erythroid maturation.TPO alone was sufficient for cell survival and proliferation in serum-depleted medium. In the absence of other growth factors, almost all CD34 + cells differentiated along the megakaryocytic pathway within 12 days. Concomitantly, the progenitor cells gradually acquired the morphologic features of mature megakaryocytes. After exposure to TPO for one week, 50% of the cells

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“…In serum-free culture medium, both CFU-GM and BFUE can be expanded from CD34+ cells in contrast to only CFU-GM in animal serum-containing medium (26).…”

Section: Discussionmentioning

confidence: 99%

Neutrophil Maturation of CD34+ Cells from Peripheral Blood and Bone Marrow in Serum-Free Culture Medium with PIXY321 and Granulocyte-Colony Stimulating Factor (G-CSF)

Smith

Bender

Berger

et al. 1997

Journal of Hematotherapy

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Bone marrow (BM) or peripheral blood (PB) CD34+ cells were cultured for 12 days in serum-free culture medium containing PIXY321 (IL-3/ GM-CSF fusion protein) with or without periodic supplements of granulocyte-colony stimulating factor (G-CSF). The cultures were evaluated at day 12 for total cell proliferation (fold increase from day 0), neutrophil differentiation by flow cytometry, using dual staining with CD15-FITC and CD11b-PE, and morphology using Wright-Giemsa and granule staining. In cultures containing PIXY321 where 6000 U/ml of G-CSF was added days 0 and 6, there was no significant difference (p > or = 0.05) in cell proliferation or the percent of CD15+/CD11b+ cells when compared with cultures with PIXY321 alone. ELISA analysis showed G-CSF levels had declined by 90% after 3 days of culture. Further studies were performed to assess the benefit of supplementing lower concentrations of G-CSF (600 U/ml) at more frequent intervals. A significant increase (p < or = 0.05) in cell proliferation and percent CD15+/CD11b+ was observed when G-CSF was added on days 0, 3, 6, and 9 (every 3 days) as compared with those cultures with PIXY321 alone. CD34+ cell proliferation without G-CSF was 19.6 +/- 4.8-fold, with G-CSF added on days 0 and 6 was 28.7 +/- 6.4-fold, and with G-CSF added on days 0, 3, 6, and 9 was 45.9 +/- 10.6-fold. Percent of CD15+/CD11b+ cells was 19.0 +/- 4.6%, 38.2 +/- 7.2%, and 58.5 +/- 6.5%, respectively, in these cultures. We observed more CD15+/CD11b+ cells, myelocytes/metamyelocytes, and secondary granule staining in cultures with G-CSF added on day, 0, 3, 6, and 9 as compared with cultures with G-CSF added on days 0 and 6 or no G-CSF added. We conclude that PIXY321 and G-CSF act synergistically on the in vitro proliferation and neutrophil differentiation of BM and PB CD34+ cells and that frequent supplements of G-CSF facilitate neutrophil differentiation.

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Direct Isolation and Expansion of Human CD34⁺Hematopoietic Stem Cells

Cited by 9 publications

References 4 publications

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34⁺ Bone Marrow and Peripheral Blood Stem Cells

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34⁺ Bone Marrow and Peripheral Blood Stem Cells

Effects of Recombinant Human Thrombopoietin Alone and in Combination with Erythropoietin and Early‐Acting Cytokines on Human Mobilized Purified CD34 ⁺ Progenitor Cells Cultured in Serum‐Depleted Medium

Neutrophil Maturation of CD34+ Cells from Peripheral Blood and Bone Marrow in Serum-Free Culture Medium with PIXY321 and Granulocyte-Colony Stimulating Factor (G-CSF)

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Direct Isolation and Expansion of Human CD34+Hematopoietic Stem Cells

Cited by 9 publications

References 4 publications

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34+ Bone Marrow and Peripheral Blood Stem Cells

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34+ Bone Marrow and Peripheral Blood Stem Cells

Effects of Recombinant Human Thrombopoietin Alone and in Combination with Erythropoietin and Early‐Acting Cytokines on Human Mobilized Purified CD34 + Progenitor Cells Cultured in Serum‐Depleted Medium

Neutrophil Maturation of CD34+ Cells from Peripheral Blood and Bone Marrow in Serum-Free Culture Medium with PIXY321 and Granulocyte-Colony Stimulating Factor (G-CSF)

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Direct Isolation and Expansion of Human CD34⁺Hematopoietic Stem Cells

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34⁺ Bone Marrow and Peripheral Blood Stem Cells

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34⁺ Bone Marrow and Peripheral Blood Stem Cells

Effects of Recombinant Human Thrombopoietin Alone and in Combination with Erythropoietin and Early‐Acting Cytokines on Human Mobilized Purified CD34 ⁺ Progenitor Cells Cultured in Serum‐Depleted Medium