Isolation and characterization of CD34+ hematopoietic stem cells from human peripheral blood by high-gradient magnetic cell sorting.

CD34+ cells, present at a frequency of 0.18 +/- 0.052% among leukocytes from peripheral blood (PB), can be rapidly and efficiently (recovery of 39.0-74.0%) enriched to a frequency of 38.6-87.1% (54.4 +/- 12.3%) by high-gradient magnetic cell separation (MACS) for immunophenotyping, characterization in colony-forming cell (CFU) assays, and further purification to homogeneity (> 98%) by multiparameter fluorescence-activated cell sorting (FACS). Enriching PB-CD34+ cells for immunophenotyping allows the detection of small subpopulations, expressing the B-cell antigens CD10, CD19, and CD20, the T-cell antigens CD45RA and CD7, and a small subpopulation expressing high levels of CD34 (1.20 +/- 0.12%), which mostly coexpress CD19 (91.9 +/- 9.05%), CD20 (64.8 +/- 14.4%), and CD38 (84.5 +/- 10.3%). All PB-CD34+ cells express elevated levels of CD71 (transferrin receptor), with a subpopulation of high expressing cells, and CD38. Some cells express CD33. MACS-enriched PB-CD34+ cells show "normal" hematopoietic colony formation in vitro. The ease and efficiency of purification of large numbers of CD34+ cells from PB by MACS is not only relevant for the characterization of migrating stem cells but also opens new possibilities for stem cell transplantation and genetic manipulation of the hematopoietic system.

[1]  Andreas Radbruch,et al.  High gradient magnetic cell separation with MACS. , 1990, Cytometry.

[2]  L. To,et al.  Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry. , 1991, Blood.

[3]  P. Lipsky,et al.  The immunosuppressive activity of L-leucyl-L-leucine methyl ester: selective ablation of cytotoxic lymphocytes and monocytes. , 1986, Journal of immunology.

[4]  G. Bonadonna,et al.  Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor. , 1989, Blood.

[5]  L. Neckers,et al.  Transferrin receptor induction in mitogen-stimulated human T lymphocytes is required for DNA synthesis and cell division and is regulated by interleukin 2. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Pileri,et al.  GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR TO HARVEST CIRCULATING HAEMOPOIETIC STEM CELLS FOR AUTOTRANSPLANTATION , 1989, The Lancet.

[7]  L. Thompson.  Stem-cell gene therapy moves toward approval. , 1992, Science.

[8]  L. Picker,et al.  Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow. , 1992, Blood.

[9]  Y. Lévy,et al.  Treatment of aggressive multiple myeloma by high-dose chemotherapy and total body irradiation followed by blood stem cells autologous graft. , 1989, Blood.

[10]  L. Lasky.  The role of the laboratory in marrow manipulation. , 1991, Archives of pathology & laboratory medicine.

[11]  D. Waber,et al.  AMITRIPTYLINE THERAPY IN PATIENTS WITH ANOREXIA NERVOSA , 1976, The Lancet.

[12]  B. Asenbauer,et al.  Sequential expression of CD34 and CD33 antigens on myeloid colony‐forming cells , 1989, European journal of haematology.

[13]  D. Huhn,et al.  Phenotyping of peripheral blood hemopoietic progenitor cells – in vitro cultures using CD34‐/CD33‐immunomagnetic purging , 1991, European journal of haematology.

[14]  D. Caracciolo,et al.  Differential activity of recombinant colony‐stimulating factors in supporting proliferation of human peripheral blood and bone marrow myeloid progenitors in culture , 1989, British journal of haematology.

[15]  B. Bostrom,et al.  CLINICAL COLLECTION AND USE OF PERIPHERAL BLOOD STEM CELLS IN PEDIATRIC PATIENTS , 1989, Transplantation.

[16]  D. Huhn,et al.  Multiparameter flow‐cytometrical quantitation of circulating CD34+‐cells: correlation to the quantitation of circulating haemopoietic progenitor cells by in vitro colony‐assay , 1991, British journal of haematology.

[17]  I. Bernstein,et al.  Positive selection of hematopoietic progenitors from marrow and peripheral blood for transplantation. , 1990, Journal of clinical apheresis.

[18]  D. Weisenburger,et al.  Autologous peripheral hematopoietic stem cell transplantation restores hematopoietic function following marrow ablative therapy. , 1988, Blood.

[19]  H. Nakauchi,et al.  Colony formation of clone-sorted human hematopoietic progenitors. , 1990, Blood.

[20]  I. Bernstein,et al.  Antigen CD34+ marrow cells engraft lethally irradiated baboons. , 1988, The Journal of clinical investigation.

[21]  C. Eaves,et al.  Selective expression of CD45 isoforms on functional subpopulations of CD34+ hemopoietic cells from human bone marrow , 1990, The Journal of experimental medicine.

[22]  P. Stiff,et al.  Autologous stem cell transplantation using peripheral blood stem cells. , 1987, Transplantation.

[23]  A. Zander,et al.  Transplantation with blood stem cells. , 1991, Blood cells.

[24]  D. Weisenburger,et al.  High-dose therapy and autologous peripheral blood stem cell transplantation for patients with lymphoma. , 1989, Blood.

[25]  E. Shimizu,et al.  Effects of recombinant human G-CSF, GM-CSF, IL-3, and IL-1 alpha on the growth of purified human peripheral blood progenitors. , 1990, Blood.

[26]  J. Liesveld,et al.  Maturation-dependent adhesion of human B cell precursors to the bone marrow microenvironment. , 1990, Journal of immunology.

[27]  H. Preisler,et al.  Isolation and characterization of the CD34+ hematopoietic progenitor cells from the peripheral blood of patients with chronic myeloid leukemia. , 1991, International journal of cell cloning.

[28]  C. Eaves,et al.  Increased erythropoietin-receptor expression on CD34-positive bone marrow cells from patients with chronic myeloid leukemia , 1992 .

[29]  T. Miyawaki,et al.  Antagonistic effects of interleukin 6 and G-CSF in the later stage of human granulopoiesis in vitro. , 1990, Experimental hematology.