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We have examined the capacity of highly purified human CD34+ marrow cell isolates from unrelated, HLA-mismatched donors to establish in vitro hematopoiesis on recipient marrow stromal cells in 2-stage hematopoietic long-term marrow cultures (H-LTMC). HLA-typing of both peripheral blood mononuclear cells and CD34+ marrow cells was performed for both HLA class I and HLA class II antigens for eight healthy individuals. Significant antigenic mismatches for these molecules ranged from three to six antigens for each recipient-donor pair. Comparison of MHC antigen expression by peripheral blood cells and CD34+ marrow cell isolates confirmed the presence of identical HLA-A, -B, and -C, and -DR specificities on the surface of these cells. Typing of -DQ specificities, however, was not consistently reactive on CD34+ cells. The > or = 20% plating efficiency of purified CD34+ cells for BFU-E, CFU-GM, and CFU-MIX allowed us to use inoculum doses of 10(3), 10(4), and 10(5) cells to determine the efficiency of allogeneic CD34+ cells in achieving in vitro engraftment and the establishment of hematopoiesis in H-LTMC. Engraftment of adherent BFU-E, CFU-GM, and CFU-MIX was equally efficient for autologous and allogeneic CD34+ cells. In vitro hematopoiesis reflected by the cumulative recoveries of progenitor cells over time was also equivalent for allogeneic and autologous CD34+ cells. These results demonstrate that highly purified, HLA-mismatched CD34+ marrow cells proliferate and establish in vitro hematopoiesis as efficiently as autologous cells in marrow derived stromal cell cultures and confirm that interactions between stromal cells and highly purified CD34+, DR-, and CD34+, DR+ marrow cell isolates are not MHC-restricted.

This report presents the results of studies using long-term bone marrow cultures (LTBMC) of human bone marrow cells to investigate the effect of HIV-1 on in vitro hematopoiesis. Confluent stromal cell layers established from human bone marrow cells were irradiated to eliminate residual hematopoietic progenitor cells and exposed to HIV-1ADA or to HIV-1IIIB, monocytotropic and lymphocytotropic strains of HIV-1, respectively. A productive infection did not develop in cultures exposed to HIV-1IIIB but did for cultures exposed to HIV-1ADA as there was a progressive increase in HIV-1 p24 antigen. Stromal cell layers infected with HIV-1ADA were also cocultured with autologous CD34+ bone marrow cells. Four days, 1, 2, and 3 weeks later, the number of colony-forming units granulocyte/macrophage (CFU-GM) in non- and HIV-infected LTBMC was determined. The number of CFU-GM increased during the first week in both non- and HIV-infected LTBMC. One week after the coculture of CD34+ cells with stromal cell layers infected with HIV-1ADA, the number of CFU-GM in six out of eight experiments was reduced compared to noninfected control LTBMC. In those six experiments, the number of CFU-GM was 53 +/- 6% standard error of the mean (SEM) of the number in noninfected LTBMC. A reduced number of CFU-GM was observed in the nonadherent fraction of HIV-infected LTBMC for at least 2 weeks. These results demonstrate that some cells in the stromal cell layers of LTBMC were targets for HIV-1 and that HIV-infected stromal cell layers suppressed or delayed the production of CFU-GM.

Human marrow cells that express the CD34 antigen but lack CD33 are able to initiate sustained, multilineage in vitro hematopoiesis in long-term Dexter cultures and are believed to include the primitive stem cells responsible for effecting long-term hematopoietic reconstitution in vivo following marrow transplantation. In studies described in this report we investigated the effects of a novel anti-CD33 immunotoxin on the clonogenic potential of normal human CD34+ marrow cells and on the ability of these cells to initiate hematopoiesis in two-stage Dexter cultures (long-term marrow cultures, LTMC). This immunotoxin (anti-CD33-bR), shown previously to kill both clonogenic myelogenous leukemia cells and normal mature myeloid progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM), consists of an anti-CD33 monoclonal antibody conjugated to purified ricin that has been modified by blocking the carbohydrate binding domains of the ricin B-chain to eliminate nonspecific binding. For our studies, normal CD34+ human marrow cells were isolated from the light-density (less than 1.070 g/ml) cells of aspirated marrow by positive selection with immunomagnetic beads linked to the monoclonal antibody K6.1. These cell isolates were highly enriched with both multipotential and lineage-restricted clonogenic, hematopoietic progenitors (mixed lineage colony-forming units, CFU-Mix; CFU-GM; and erythroid burst-forming units, BFU-E) which constituted greater than or equal to 20% of the cells. Recovery of clonogenic progenitors from these CD34+ cell preparations, following treatment with anti-CD33-bR (10 nM), was reduced by greater than or equal to 85% for CFU-GM and 20%-40% for CFU-Mix and BFU-E. However, the capacity of these cells to initiate hematopoietic LTMC was preserved. Indeed, the production of high proliferative potential (HPP) CFU-GM, BFU-E, and CFU-Mix in cultures seeded with 10(5) anti-CD33-bR-treated CD34+ marrow cells was substantially greater than that observed in LTMC seeded with equivalent numbers of untreated CD34+ cells. Moreover, concentrations of long-term culture initiating cells in CD34+ cell isolates, quantified by a limiting dilution technique, were found to be increased following anti-CD33-bR treatment. These findings support the potential usefulness of anti-CD33-bR for in vitro marrow purging or in vivo treatment to eliminate CD33+ leukemic clones, while sparing normal CD34+/CD33- stem cells that support normal hematopoiesis and hematopoietic reconstitution in vivo.