Isolation of CD34+ cells from peripheral blood and bone marrow of Tursiops truncatus.

摘要

A female bottlenose dolphin died after stranding on Tregunc beach, Brittany, France on 28 September 2010. The animal was brought into the laboratory and autopsied. Within 4 h after discovery of the animal, ~100 ml of peripheral blood were collected near the left front flipper and 10 ml bone marrow was extracted from the inside part of the left lower jaw. Mononuclear cells were purified based on density. Half the volume of lymphocyte separation medium was added to both peripheral blood and bone marrow samples, mixed and centrifuged. Mononuclear cells were removed by pipetting, pooled in a new tube, and again centrifuged. After incubation, the magnetic labelled CD34+ cells were retained in a column placed in a magnetic field, and then eluted. 3x103 CD34+ cells were isolated from the bone marrow mononuclear cells and 4x104 CD34+ from the peripheral blood cells. Yields of CD34+ cell isolation were 0.22 and 7.5% for peripheral blood and bone marrow, respectively. Then, the bottlenose dolphin CD34+ cells were cultivated in conditions similar to the proliferation of human CD34+ cells. During the proliferation phase, human CD34+ cells also underwent a partial differentiation, which could be reflected in the disappearance of the CD34+ marker at the cell surface. A flow cytometry analysis to determine the continued presence of the CD34 marker on the surface of the cells and the size and granularity of the cells was used. CD34+ cells were labelled using the FITC-coupled anti-human CD34 antibody for human CD34 cells: briefly, after washing, the cells were incubated for 30 min at 4 degrees C in PBS-FBS, in the presence of FITC-conjugated anti-CD34 antibody. FITC-conjugated anti-CD34 fluorescence (FL1-H) was collected through a 530/30 nm bandpass filter. Parameter list-mode files of 10 000 events were collected from each sample and results were expressed as a percentage of positive cells. Isotypic control labelling was performed previously, and fluorescence quadrants were set using the mouse IgG1-isotype negative control antibody. The percentages of cells still presenting a CD34+ phenotype revealed that 12% of the cells were derived from the bone marrow, and 22% of the cells were derived from peripheral blood were CD34+. Cells derived from peripheral blood appeared more heterogeneous than the ones derived from bone marrow. It is concluded that bottlenose dolphin CD34+ cells can be isolated and cultivated from peripheral blood and bone marrow using technical approaches derived from human and mammalian models