Ex vivo expansion and long-term hematopoietic reconstitution ability of sorted CD34+CD59+ cells from patients with paroxysmal nocturnal hemoglobinuria

Abstract

Autologous bone marrow transplantation (ABMT) for paroxysmal nocturnal hemoglobinuria (PNH) remains difficult so far. To expand residual normal CD34(+)CD59(+) cells isolated from patients with PNH and observe the long-term hematopoietic reconstruction ability of the expanded cells both ex vivo and in vivo, CD34(+)CD59(+) cells from 13 PNH patients and CD34(+) cells from 11 normal controls were separated from bone marrow mononuclear cells first by immunomagnetic microbeads and then by flow cytometry autoclone sorting. The cells were then cultivated under different conditions. The long-term hematopoietic supporting ability of expanded CD34(+)CD59(+) cells was evaluated by long-term culture in semi-solid medium in vitro and long-term engraftment in irradiated severe combined immunodeficiency (SCID) mice in vivo. The best combination of hematopoietic growth factors for ex vivo expansion was SCF + IL-3 + IL-6 + FL + Tpo + Epo. The most suitable time for harvest was on day 7. CD34(+)CD59(+) PNH cells retained strong colony-forming capacity even after expansion. The survival rate, complete blood cell count recovery on day 90, and human CD45 expression in different organs were similar between the irradiated SCID mice transplanted with expanded CD34(+)CD59(+) PNH cells and those with normal CD34(+) cells (P > 0.05) both in primary and secondary transplantation. These data provided a new potential way of managing PNH with ABMT.

Allogeneic Hematopoietic Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria

“…Another novel theoretically interesting approach is related to selection of normal circulating CD34+/CD59+ cells with flow cytometry from PNH patients who have a mixed population of normal and abnormal cells for the purpose of performing autologous hematopoietic cell transplantation (AHCT). As yet, no case of AHCT for PNH has been reported (Xiao et al 2010). …”