The early stages of lymphoid cell formation were studied by testing the differentiative potential of phenotypically defined subsets of CD34+ bone marrow cells. A subpopulation of CD34+ Lin- CD45RA+ cells expressing CD10 was isolated by flow cytometry. Such cells are CD38+, HLA-DR+, do not express significant levels of Thy-1 and c-kit, lack erythroid, myeloid, megakaryocytic potential, and give rise only to lymphoid T, B, natural killer (NK), and dendritic cells (DC) in kinetics and titration experiments. Limiting dilution analysis demonstrates the existence of multipotential B/NK/DC progenitor clones in the CD34hi Lin-CD10+ adult bone marrow cell population. Thus, nonprimitive progenitors for lymphoid cells and for DCs can be distinct from those of myeloid, megakaryocytic, and erythroid cells, implying that the DC lineage is developmentally more closely related to the lymphoid lineage than to the myeloid lineage. This study provides new insights into the organization and development of the human lympho-hematopoietic system.

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin-ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA-cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.

Summary. Haemopoietic cytopenias are a frequent occurrence in human immunodeficiency virus type‐1 (HIV‐1) induced disease. In order to examine the possible direct inhibition of marrow haemopoiesis by HIV‐1, we have investigated the effect of HIV‐1 infection on myelopoiesis in long‐term bone marrow cultures. In vitro exposure of normal marrow cultures to three different lymphocytotropic HIV‐1 isolates resulted in productive infection, as demonstrated by a progressive increase of gag p24 antigen. In these experiments, ICR‐3 isolate, but not LAV' or NL4–3 isolates, accelerated the loss of non‐adherent cells. A differential ability of these HIV‐1 isolates to suppress myelopoiesis was confirmed in long‐term cultures in which virus was added continuously. In these cultures, ICR‐3, and to a lesser extent also NL4–3, but not LAV', induced a progressive decrease in the number of total non‐adherent cells as well as non‐adherent colony forming units‐granulocyte/macrophage (CFU‐GM). Furthermore, exposure of normal purified CD34+ cells to ICR‐3 induced defects in their ability to form haemopoietic colonies: this inhibitory effect was significantly relieved by pretreatment of ICR‐3 with an anti‐gp 120 antibody. Similar exposure of CD34+ cells to LAV' and NL4–3 induced no such defects. These data indicate that some HIV‐1 isolates can impair bone marrow haemopoiesis in a dose‐dependent fashion, acting, at least in part, at the level of haemopoietic stem/ progenitor cells.

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.

The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.