ABSTRACT
Paroxysmal nocturnal hemoglobinuria [PNH], a clonal hematopoietic stem cell disorder, manifests when the PNH clone populates in the hematopoietic compartment. We explored the roles of different apoptosis of GPI+ and GPI- [glycosylphosphatidylinositol] cells and CD8+ lymphocytes in a selection of PNH clones. Granulocytes from PNH patients and normal controls were subjected to an apoptosis assay using annexin V. Hematopoietic cell in semisolid media were cultured with or without CD8+ lymphocytes. In PNH, CD59+ granulocytes exhibited more apoptosis than their CD59- counterparts, after 0 or 4 hours in liquid growth culture system [mean [standard error of mean]: 2.1 [0.5] vs 1.2 [0.2], P=.01 at 0 hour and 3.4 [0.7] vs 1.8 [0.3], P=.03 at 4 hour, respectively]. The presence of mononuclear cells [MNCs] rendered a greater difference in apoptosis. The percentages of apoptotic CD59+ granulocytes measured at 4 hours with or without MNC fraction were correlated with the sizes of PNH clones [r=0.633, P=.011; and r=0.648, P=.009; respectively]. The autologous CD8+ lymphocytes inhibited CFU-GM and BFU-E colony formation in PNH patients when compared with normal controls [mean [SEM] of percentages of inhibition: 61.7 [10.4] vs 11.9 [2.0], P=.008 for CFU-GM and 26.1 [6.9] vs 4.9 [1.0], P=.037 for BFU-E]. Increased apoptosis of GPI+ blood cells is likely to be responsible in selection and expansion of PNH clones. MNCs or possibly CD8+ lymphocytes may play a role in this phenomenon
ABSTRACT
Introduction: Type 2 diabetes, caused by an insulin resistant, is associated with endothelial cell dysfunction and impaired vascular homeostasis, resulting in vascular inflammation and atherosclerosis. Recently, endothelial progenitor cells (EPCs) which originated from the bone marrow are considered to be an important regulator in vascular homeostasis and a surrogate marker for vascular complications. According to this, EPC dysfunction may play an important role in causing the diabetic-associated vascular complications. Objective: To compare number and characteristic of EPCs in Type 2 diabetic patients with those of normal healthy subjects. Methods: The number of EPCs from twenty Type 2 diabetic patients and twenty healthy subjects was studied by using flow cytometry. The isolated EPCs were cultured and characterized by Dil-AcLDL engulfment. The various glucose concentrations were employed in the EPC culture in order to determine the effect of hyperglycemia on the number and viability of the EPCs. Results: The number of EPCs in Type 2 diabetic patients was significantly decreased compared to healthy subjects (5.5 \× 10⁶ \± 0.5 \× 10⁶ vs. 23 \× 10⁶ \± 2.3 x 10⁶; P \< 0.01). There was an inverse correlation between the EPC numbers and the concentrations of plasma glucose (r = -0.045) as well as HbA1c (r = -0.336). The culture of EPCs from diabetic patients took a significantly longer period of time to develop mature EPCs than control (30.8 \± 3.9 days vs. 22.4 \± 2.7 days, P \< 0.01). In addition, the number of cultured EPCs was significantly reduced when the glucose concentration in culture medium was greater than 16.5 mmol/l (P \< 0.05). These results demonstrated that glucose has a negative effect on the number and viability of EPCs in a dose-dependent fashion. Conclusion Hyperglycemic condition in Type 2 diabetes has a negative effect on the number and viability of circulating EPCs in a dose-dependent fashion.
ABSTRACT
Objective: To isolate and characterize mesenchymal stem cells (MSCs) from peripheral blood and G-CSF mobilized peripheral blood. Methods: Mononuclear cells (MNCs) were isolated from peripheral blood, G-CSF mobilized peripheral blood and bone marrow using gradient centrifugation. The numbers of MSCs in these three sources were quantified using flow cytometry. The isolated MNCs were then cultured to generate MSCs. The MSCs generated from those three sources were studied in term of the MSC marker (CD73, CD90, CD105 and CD106) expression, the ability to generate colony (CFU-F) in culture and the ability to differentiate toward osteocyte and adipocyte-lineages. Results: The percentage of cells that expressed CD90 in fresh MNC populations isolated from bone marrow (BM-MNCs), peripheral blood (PB-MNCs) and mobilized peripheral blood (MPB-MNCs) is 1.94, 2.1 and 0.05 respectively, while the expression of CD73 in BM-MNCs, PB-MNCs and MPB-MNCs is 5.2, 15.2 and 7.8 respectively. The percentage of cells that expressed CD105 in BM-MNCs, PB-MNCs and MPB-MNCs is 5.3, 4.1 and 2.75, respectively while the expression of CD106 in those three populations is 2.82, 2.36 and 4.5 respectively. The ability of BM-MNCs, PB-MNCs and MPB-MNCs to generate colony in culture (CFU-F) is 67, 30 and 48 colonies per 10⁶ plating MNCs, respectively. After culture for three passages, more than 64% of BM-MNCs, PB-MNCs and MPB-MNCs homogeneously expressed CD73, CD90 and CD105. In contrast, the expression of CD45 (marker of hematopoietic cells) in those populations is negative. In addition, the bone marrow-derived MSCs also have an ability to differentiate toward osteocyte and adipocyte-lineages. Conclusion: We have successfully isolated and characterized MSCs from both peripheral blood and G-CSF mobilized peripheral blood. Those MSCs expressed several MSC markers, including CD73, CD90, CD105 and CD106, and able to generate colonies in culture in a manner similar to those of BM-MSCs. Our results suggest that these PB-MSCs and MPB-MSCs might be used as an alternative source for the clinical treatment in the future.