LONG-TERM CULTURE OF HUMAN HEMATOPOIETIC STEM CELLS

A PHYSIOLOGICAL THREE-DIMENSIONAL, LONG-TERM CULTURE SYSTEM HAS BEEN DEVELOPED FOR THE GROWTH OF BONE MARROW. THIS SYSTEM INVOLVES THE ESTABLISHMENT OF STROMAL CELLS ON A SUSPENDED NYLON FILTRATION SCREEN, THE GROWTH OF STROMAL CELLS TO SUBCONFLUENCE, AND THE INOCULATION OF STROMA WITH FRESH OR CRYOPRESERVED BONE MARROW CELLS. STROMAL ELEMENTS DISTRIBUTE THEMSELVES THROUGHOUT THE MESHWORK, SECRETE MATRIX PROTEINS, AND ESTABLISH AN INTRICATE MICROENVIRONMENTFOR THE SUPPORT OF HEMATOPOIESIS. ACTIVE PRODUCTION OF MYELOID AND ERYTHROID CELLS IS SEEN FOR AT LEAST 12 WEEKS. PRELIMINARY ANALYSIS OF THIS SYSTEM REVEALS THE ADHERENT ZONE TO BE ACTIVELY PROLIFERATING AND TO CONSIST OF MULTILINEAGE IMMATURE CELLS. THIS ADHERENT LAYER SUPPORTS ACTIVE PROLIFERATION OF CFU-GM, CFU-GEMM, AND ERYTHROID PROGENITOR CELLS, AS INDICATED BY STANDARD METHYLCELLULOSE AND PLASMA CLOT ASSAY SYSTEMS. MULTILINEAGE HEMATOLOGIC EXPRESSION IN ADHERENT AND NONADHERENT ZONES HAS BEEN CONFIRMED BY CYTOFLUOROGRAPHIC ANALYSIS AND STANDARD DIFFERENTIAL COUNTS. THESE FINDINGS INDICATE THAT THE THREE-DIMENSIONAL NYLON MESH SYSTEM IS A NOVEL IN VITRO METHOD THAT SUPPORTS THE GROWTH OF SEVERAL HEMATOLOGIC LINEAGES CONCURRENTLY AS WELL AS THE PROLIFERATION OF VARIOUS RAT, MONKEY, AND HUMAN BONE MARROW STEM CELLS.