Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

Human fetal bone fragments implanted subcutaneously in immunodeficient (SCID) mice maintain active human hematopoiesis. In this study, we show that this human hematopoietic microenvironment supports the engraftment and differentiation of HLA-mismatched, CD34+ primitive hematopoietic progenitor cells isolated from fetal and adult human bone marrow (BM). The BM CD34+ cells were depleted of CD2, CD14, CD15, CD16, glycophorin A, and CD19 lineage-committed cells (CD34+Lin-). Donor cell engraftment was manifested by the presence of B (CD19+) and myeloid (CD33+) cells of donor HLA phenotype. Successful engraftment was observed as early as 4 weeks after fetal BM donor cell injection and sustained for at least 12 weeks, with engraftment success rates of 100% (11/11 grafts) and 92% (11/12 grafts) at 8 and 12 weeks, respectively. Mixed BM chimerism of donor and endogenous cells was consistently observed in SCID-hu bones successfully engrafted with HLA-mismatched CD34+Lin- donor cells. Preconditioning of the SCID-hu bone with a single dose of sublethal (350 rad) whole body irradiation (WBI) immediately before cell injection enhanced the repopulation of the bone grafts with donor cells and, in some instances, resulted in complete repopulation. After WBI, as few as 500 fetal bone marrow CD34+Lin- cells injected in the human bone grafts resulted in donor-derived hematopoiesis. Donor progenitor cells recovered from the SCID-hu bone grafts 8 weeks postinjection had the capacity to repopulate secondary groups of HLA-disparate fetal human bones in SCID-hu mice with B and myeloid cells as well as CD34+ cells in some recipients. In addition, these cells repopulated fetal human thymus fragments in SCID mice with donor thymocytes including immature CD4+CD8+ and mature CD4+CD8- as well as CD4-CD8+ subsets. These results indicate that the fetal human bone implants of SCID-hu mice can support the maintenance of a cell population that has both multilineage potential and repopulating potential for periods of time as long as 16 weeks. The SCID-hu bone model consistently supported the engraftment of both fetal and adult CD34+Lin- cells without the administration of exogenous human cytokines to these animals. This model is currently being used to permit the isolation and characterization of candidate human hematopoietic stem cells (HSCs) and provide important information critical for human HSC therapy in humans.