Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, venous thrombosis, and bone marrow (BM) failure. Until now, allogeneic hematopoietic stem cell transplantation is still the only way to cure PNH. Eculizumab, although very promising, is not the eradication of the disease because of raising the possibility of severe intravascular hemolysis if therapy is interrupted. Here we enriched the residual bone marrow normal progenitor cells (marked by CD34+CD59+) from PNH patients, tried to find an effective way of expanding the progenitors cells used for autologous bone marrow transplantation (ABMT).

Objective To expand CD34+CD59+ cells isolated from patients with PNH and observe the long-term hemaotopoietic reconstruction ability of the expanded cells both ex vivo and in vivo.

Methods CD34+CD59+ cells from 13 patients with PNH and CD34+ cells from 11 normal controls were separated from the bone marrow monouclear cells first by immunomagnetic microbead and then by flow cytometry autoclone sorting. The selected cells were then cultivated under different conditions for two weeks to find out the optimal expansion factors. 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.

Results The best combination of hematopoietic growth factors for ex vivo expansion was SCF+IL-3+IL-6+FL+Tpo+Epo, and the most suitable time for harvest was on day 7. Although the CD34+CD59+ PNH cells had impaired ex vivo increase compared with normal CD34+ cells (the biggest expansion was 23.49±3.52 fold in CD34+CD59+ PNH cells and 38.82±4.32 fold in CD34+ normal cells, P<0.01 ), they remained strong colony-forming capacity even after expansion ( no difference was noticed in CFCs or LTC-IC of PNH CD34+CD59+ cells before and after expansion, P>0.05). According to the above data, 11/13(84.3%) patients with PNH can get enough CD34+CD59+cells for ABMT after expansion. The survival rate and human CD45 expression in different organs was similar between the irradiated SCID mice transplanted with expanded CD34+CD59+ PNH cells and those with normal CD34+ cells (P>0.05). The peripheral blood cell count recovered on day 90 in mice transplanted with PNH cells, which was compatible with those transplanted with normal cells (P>0.05). On secondary transplantation, the peripheral blood cell count returned to almost normal on day 30 in mice transplanted with either PNH cells or normal cells. Lower CD45 percentage was found in secondary transplantation compared with primary transplantation but no difference between mice transplanted with different cells.

Conclusion Isolated CD34+CD59+ cells from patients with PNH can be effectively expanded ex vivo and can support lasting hematopoiesis both ex vivo and in vivo. These data provide a new potential way of managing PNH with ABMT.

Disclosures: No relevant conflicts of interest to declare.

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