Long-term ability for hematopoiesis of CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria: experiment with mice

Abstract

To obtain the evidence of long-term hematopoiesis by normal clone stem cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). 10 ml fresh bone marrow was collected from 2 PNH patients. Normal bone marrow was obtained from the ribs resected during operation of 2 patients without hematopathy. CD34(+)CD59(+) cells were isolated, purified, and expended. 12 total body irradiated SCID mice were divided into 2 equal group to undergo transplantation of the expanded CD34(+)CD59(+) cells from PNH patients (experiment group 1) or normal patients (control group 1). Four mice underwent transfusion of cell-free medium as blank control group 1. 30 and 60 days after the transplantation peripheral blood samples were collected and 90 days after the transplantation heart blood samples were collected. Spleens were taken out to prepare suspension of splenocytes and bacterium-free bone marrow cell suspension was prepared from the bilateral femurs. 90 days after the primary transplantation another 16 mice underwent total body irradiation and then divided into 2 equal groups to undergo transplantation of the bone marrow cells of the mice of the experiment group 1 and control group 1 (experiment group 2 and control group 2). Another 4 mice were transfused with cell-free medium fluid (blank control group 2). The levels of blood cells of all groups were calculated 30, 60, and 90 days after the primary and 30 days after the secondary transplantation. The cell percentages of peripheral blood, spleen, and bone marrow after the primary transplantation and 30 days after the secondary transplantation were detected by flow cytometry. PCR was used to detect whether the sex determining region Y existed in the female SCID mice after transplantation. Ninety days after primary transplantation, the peripheral blood cell levels of both experiment group 1 and control group 1 recovered to normal. Expression of CD45 was detected in the mice without significant difference between the 2 groups. Thirty days after the secondary transplantation, the peripheral blood cell levels of the experiment group 2 and control group 2. Expression of CD45 was detected in both the experiment group 2 and control group 2 without significant difference between these two groups. Human SRY gene could be detected by PCR in the female SCID mice after transplantation. CD34(+)CD59(+) cells isolated from PNH patients and expanded in vitro can be successfully engrafted with long-term ability in hematopoiesis not different from that of the normal CD34(+)CD59(+) cells.