This study comparing the relative white cell (WBC)-depleting efficiency of single and double filtration used two filters and new, sensitive, and reliable methods for performing WBC counts on WBC-depleted blood products. A single filtration of red cell (RBC) concentrates with a cotton-wool filter reduced WBC content by 98.64 percent, but the range of residual WBCs was wide, and many filtered units still contained more than the theoretical immunizing dose of 5 to 10 x 10(6) WBCs. A second filtration, however, always produced RBC units that had less than 5 x 10(6) WBCs. Although the degree of WBC depletion observed after a single filtration of a 6-unit pool of random-donor platelet concentrates was greater with a polyester filter than with the cotton-wool filter (98.92 vs. 98.14% reduction, respectively, when mean prefiltration WBC count was less than 600 x 10(6], in both cases, 25 percent of filtered products still contained greater than 5 x 10(6) WBCs; a second filtration (with the cotton-wool filter), however, produced units that were always below the immunizing dose. All double-filtered platelet concentrates had less than 10(6), and one-half had less than 10(4) residual WBCs. Platelet loss was similar with both filters (+/- 16% loss with one filtration). The effectiveness of the filters in producing products that were WBC-depleted below the immunizing dose was dependent on the prefiltration WBC content (but not on the age of the units), and it may be worthwhile to employ methods to ensure that total prefiltration WBC count of the product is less than 400 x 10(6).(ABSTRACT TRUNCATED AT 250 WORDS)

New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.

Human pluripotent stem cells (hPSCs) are a promising source of autologous endothelial progenitor cells (EPCs) that can be used for the treatment of vascular diseases. However, this kind of treatment requires a large amount of EPCs. Therefore, a highly efficient, robust, and easily reproducible differentiation protocol is necessary. We present a novel serum-free differentiation protocol that exploits the synergy of multiple powerful differentiation effectors. Our protocol follows the proper physiological pathway by differentiating EPCs from hPSCs in three phases that mimic in vivo embryonic vascular development. Specifically, hPSCs are differentiated into (i) primitive streak, which is subsequently turned into (ii) mesoderm, which finally differentiates into (iii) EPCs. This differentiation process yields up to 15 differentiated cells per seeded hPSC in 5 days. Endothelial progenitor cells constitute up to 97% of these derived cells. The experiments were performed on the human embryonic stem cell line H9 and six human induced pluripotent stem cell lines generated in our laboratory. Therefore, robustness was verified using many hPSC lines. Two previously established protocols were also adapted and compared to our synergistic three-phase protocol. Increased efficiency and decreased variability were observed for our differentiation protocol in comparison to the other tested protocols. Furthermore, EPCs derived from hPSCs by our protocol expressed the highproliferative-potential EPC marker CD157 on their surface in addition to the standard EPC surface markers CD31, CD144, CD34, KDR, and CXCR4. Our protocol enables efficient fully defined production of autologous endothelial progenitors for research and clinical applications.