Methods comprising apoptosis inhibitors for the generation of transgenic pigs

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Stem Cell Related Patent Number US6960473

Title:In vitro mass production of human erythroid cells from the blood of normal donors and thalassemic patients
Inventors:Migliaccio, Giovanni; Rome, Italy
Summary:Herein is described a system of producing primary human erythroid cells by culturing light-density cells in a first culture medium for the proliferation of cells, and re-culturing the cells in a second culture medium for the differentiation of cells into the erythroid cells. This invention relates to a novel two-step culture method for mass production in vitro of erythroid cells from either CD34+ (105 cells/mL) or light-density (106 cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method provided allows for the culturing of cells in the presence of dexamethasone and estradiol (10-6 M each) and the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). Further described are the generation of approximately 1-2x107 erythroblasts in the proliferative phase for each milliliter of blood collected from normal donors or thalassemic patients, such that the compositions exhibited 90% of CD45low/glycophorin (GPA)neg/CD71low cells at day 7, 50-60% of which became CD45neg/GPA+/CD71high by days 15-20. Also provided are data relating to cells from days 7 to 12 of the proliferative phase which were transferred in a differentiation medium containing EPO and insulin, and which progressed to mature erythroblasts (>90% benzidinepos and CD45neg/GPA+/CD71medium) in 4 days. Also offered are applications of such a method, which, due to the high number of erythroid cells that are generated from modest volumes of blood, should find uses in donor-specific studies of erythroid differentiation.
Abstract:We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34+ (105 cells/mL) or light-density (106 cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10−6 M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated about 1-2107 erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45low/glycophorin (GPA)neg/CD71low cells at day 7, 50-60% of which became CD45neg/GPA+/CD71high by days 15-20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (>90% benzidinepos and CD45neg/GPA+/CD71medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.
US Patent Website:Click Here for Full Text of Patent
Title Number:US6960473
Application Number:US2004000786461
Date Filed:26/02/2004
Date Published:01/11/2005
Assignee:Istituto Superiore di Sanita, Rome, Italy


 
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