HARVESTING, CHARACTERIZATION, AND CULTURE OF CD34(-MARROW, PERIPHERAL-BLOOD, AND CORD-BLOOD() CELLS FROM HUMAN BONE)

Stem and progenitor cells from a variety of sources including bone mar row, cord blood, and peripheral blood have been used for transplantati on. This study compares CD34 cells from all three sources. Flow cytome try analysis of CD34 cells in multiple samples of normal peripheral bl ood and patient peripheral blood mobilized with chemotherapy (cyclopho sphamide/VP16), chemotherapy plus granulocyte colony stimulating facto r (G-CSF), and G-CSF alone were compared to bone marrow and cord blood . Although the relative distribution of CD34 percentages in each prepa ration of cells varied widely, on average the percentage of CD34 cells in these different preparations was 0.15%, 0.6%, 2%, 0.45%, 1.68%, an d 0.83% respectively. CD34 subset analysis was performed on these cell preparations using multicolor flow cytometry and antibodies to CD33, CD13, CD45RA, CD19, CD71, and CD38. The major differences observed wer e that bone marrow CD34 cells contain high percentages of CD19(+) cell s not found in significant quantity in the other cell preparations and cord blood CD34 cells contained a higher percentage of CD38-cells tha n the other cell preparations. A magnetic bead system was used with an ti-CD34 antibody to purify CD34 cells from mobilized peripheral blood apheresis products, cord blood, and bone marrow. Efficient selection w ith high purities of CD34 cells was achieved with each of the cell pre parations. Comparison of colony-forming activity of each of the cell p reparations showed cord blood and mobilized peripheral blood to have s lightly higher cloning efficiencies than bone marrow with higher numbe rs of erythroid blast-forming units (BFU-E) also observed in cord bloo d CD34 cells. Culture of isolated CD34 cells in liquid culture with in terleukin-3, stem cell factor, G-CSF, and granulocyte-macrophage GM-CS F showed over a 100-fold expansion in cell numbers after 25 days, with the peak expansion of colony-forming cells occurring between days 11 and 16. Analysis of day-10 cells from these cultures showed them to be predominantly promyelocytes, myelocytes, and metamyelocytes, with cor d blood CD34 cultures showing more promyelocytes than peripheral blood or bone marrow and bone marrow showing more metamyelocytes, Compariso n of the proliferation of CD34 cells from these different cell prepara tions showed that cord blood CD34 cells cultured for 10 days averaged an 85-fold increase in cell numbers followed by mobilized peripheral b lood CD34 cells, with an average 56-fold increase, and bone marrow CD3 4 cells, with an average 49-fold increase.