T cell differentiation of CD34+ stem cells in cultured thymic epithelial fragments

A process of coculturing Allogeneic Cultured Thymic Epithelial Fragments and bone-marrow derived stem cells from HIV seropositive patients whereby the stem cells are differentiated into immunocompetent T cells free of HIV infection, comprising the following steps:

• establishing cultured thymic epithelial fragment cultures by:
• excising thymus tissue as a metabolic specimen avoiding mechanical pressure and heat cauterization and placing the tissue in chilled media,
• removing the thymic capsule from the excised thymic tissue, mincing the tissue into small fragments and agitating the fragments in complete media to wash out thymocytes,
• depleting thymocytes and hematopoietic cells including dendritic cells by incubating the fragments on sterile tissue rafts which are partially immersed in complete medium supplemented with 2'-deoxyguanosine, at about 37.degree. C. in a partial CO.sub.2 atmosphere for at least seven days, and
• culturing the T cell depleted fragments to optimize stromal viability in complete medium supplemented with hydrocortisone, epidermal growth factor, cholera enterotoxin, insulin, adenine, and sodium pyruvate,
• isolating CD34+ stem cells from HIV seropositive patients by:
• collecting bone marrow from aspirates in Heparinized tubes,
• performing Ficoll-Hypaque density centrifugation to obtain nucleated bone marrow cells from bone marrow aspirates diluted in normal saline,
• depleting CD2+ lymphocytes by rosette formation with sheep Red Blood Cells treated with 2-amino-ethyl isothiouronium bromide, followed by a second Ficoll-Hypaque density centrifugation,
• removing macrophage/monocytic cells by adherence on plastic Petri dishes,
• removing non-CD34 cells by culture with monoclonal antibodies or immunomagnetic microbeads, followed by separation over a monoclonal antibody-coated panning flask or magnetic column, and
• coculturing the Cultured Thymic Epithelial Fragments and purified CD34+CD38- Stem Cells by:
• seeding a minimum of 5,000 lineage negative stem cells or 50,000 lineage committed stem cells by infusion onto the surface of the Cultured Thymic Epithelial Fragments in transwell plates in Iscove's/Ham's medium containing IL-2 at about 37.degree. C. in a partial CO.sub.2 atmosphere until differentiated into mature T cells, replacing medium every few days, and
• isolating the differentiated T cells by aspirating the cells from the culture wells, washing, resuspending in saline, subjecting the cells to Ficoll-Hypaque density centrifugation to remove dead cells, washing the mononuclear cells, and
• resuspending the pure mononuclear cells in saline.