The objectives of this proposal are to establish the conditions necessary for the long-term culture and expansion of human hematopoietic progenitor cells, to develop in vitro and in vivo assay systems for such clonogenic precursors, and to develop monoclonal antibodies for their identification and purification. Establishment of long-term cultures will be initiated using a variety of procedures. Culture conditions continuously generating nonadherent hematopoietic cells will be used to develop an assay for clonogenic hematolymphoid progenitor cells. Eventual isolation of human hematopoietic stem cells will depend upon the identification of cell surface antigens which mark cells with increased progenitor activity. Known mAb's (CD34 and Thy 1) and newly generated mAb's will be tested for their ability to mark either pluripotent stem cells or more committed progenitor populations. Bone marrow cells will be sorted with these mAb's and tested in the assays detailed within to determine the frequency of responding cells and their proliferation/differentiation potential. Introduction of HLA-disparate cells into a SCID-hu mouse results in human lymphoid reconstitution and thus should serve as an in vivo assay system for multipotent and/or restricted precursor cells. The feasibility of using the SCID-hu mouse as an in vivo assay will be examined extensively. The availability of simultaneous long-term in vitro and in vivo systems for the characterization and purification of human progenitor cells will allow a more precise dissection of the defects observed in various human immunologic deficiencies.
