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Paroxysmal nocturnal hemoglobinuria hematopoietic stem / progenitor cells in vitro single cell culture studies and matrix Preliminary Study

Author: HanBing
Tutor: WuYongJi
School: Peking Union Medical College , China
Course: Internal Medicine
Keywords: Progenitor cells Normal human serum Hematopoietic stem cells Cell expansion Single cells Phenotypic Pancytopenia Medical Human peripheral blood Bone marrow stromal cells
CLC: R55
Type: PhD thesis
Year: 2000
Downloads: 34
Quote: 0
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Abstract

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a syndrome characterized by intravascular hemolysis, severe anemia and venous thrombosis. PNH frequently occurs in association with suppressed hematopoiesis, including frank aplastic anemia (AA). It is known that PNH patients have a somatic mutation of the X-linked PIG-A gene which occurs on a hematopoietic stem cell level. This results in a proportion of blood cells being deficient in all glycosylphosphatidylinositol (GPI)-anchored surface proteins. Although these GPI-deficient cells can explain many of the clinical symptoms, including hemolysis mediated by complement and venous thrombosis, the pathogenesis of PNH is still somewhat obscure and many questions remain. The present study was designed to determine whether the abnormal (GPI-) clones had growth advantage over the normal (GPI+) ones. Besides, the influence of sera and stromata from PNH patients on the growth of the hematopoietic progenitor cells was also tested. The research may be helpful further to elucidate the pathogenesis of PNH.1 CD59 was chosen as a marker of the PNH phenotype based on its high and homogeneous expression on fluorescent staining. The CD34 and CD59 expressions in peripheral blood and bone marrow samples from 21 PNH patients and 8 normal volunteers were analyzed by fiowcytometor. The results were as follows: 1) While the number of CD34+ cells in both peripheral blood and bone marrow in patients with clinical remission had no significant difference from that in normal controls, it decreased remarkably in patients who had clinical synptoms. 2) The CD34+ cells in peripheral blood of the patients exhibited predominantly normal CD59 phenotype, which had no relationship either to the patients’ clinical manifestations or to the CD59 expressions on bone marrow CD34+ cells. 3) Most of the CD34+cells in bone marrow lacked CD59 antigen in patients who had recently hemolytic attack, but showed nearly normal CD59 expression in patients with clinical remission.2 To investigate the ability of the stroma-independent growth, multilineage differentiation and progenitor cell expansion, the CD34+CD59+ and CD34+CD59- cells from PNH patients and CD34+CD59+ cells from normal volunteers were sorted as single cell into 96-well flat-bottom culture plates containing culture medium supplemented with SCF、 IL-3、 EPO、 GM-CSF、 G-CSF、 IL-6n TPO and FLt-3 ligand. The following results were obtained:1) i) While single PNH CD34+CD59-cells had higher capacity for plating efficiency, colony(cells≥50) formation and cell expansion than the PNH CD34+CD59+ cells, their capacity of large colony (cells≥500) formation, average cell production and secondary colony formation was nearly the same, which suggested a relative growth predominance of the CD34+CD59-clone. ii) Multi-cell culture had a better cell expansion than single cell culture.2) i) Both the single CD34+CD59- cells from PNH patients and the single CD34+CD59+ cells from normal controls had similar cell plating efficiency, colony and large colony formation.The CD34+CD59-cells had lower average cell production and cell expansion capacity, ii) The CD34+CD59- clone showed a diminished secondary colony formation.3) i) The single CD34+CD59+ cells from both PNH patients and normal controls showed the same cell plating efficiency and colony formation. The PNH CD34+CD59+ cells exhibited much lower capacity for large colony formation, average cell production and total cell expansion.

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