Culture of hematopoietic stem cells purified from murine bone marrow.

The results of the Y-chromosome in situ hybridization experiments, the MRA assessment, and the long-term production of CFU-GM in vitro indicate that our protocol to sort low density WGA+, 15/1.1-, Rh123 dull cells enriches about 200-fold for PHSC. Assays for spleen colony formation (CFU-S) and radioprotection (30-day survival) were shown to be unspecific for PHSC, and, therefore, we lack a quantitative PHSC assay. The absolute number of PHSC in the bone marrow is not known any more, the purity of our sorted population likewise is unknown. Long-term repopulating cells (PHSC) could be separated from short-term repopulating ones by using Rh123 staining. The short-term repopulating cells (Rh123 bright) provided sufficient offspring to protect lethally irradiated mice until endogenous PHSC could reconstitute hematopoiesis. These cells are therefore of interest for bone marrow transplantation, because they provide radioprotection without long-term repopulation and graft-versus-host disease. For gene therapy these cells are of limited use, and PHSC with extensive replication are needed. The PHSC were not cultured successfully. Less than 15% of the sorted Rh123 dull cells responded in semisolid or liquid cultures in the presence of growth factors. Proliferation without differentiation was not observed. This may indicate that the right growth factor has not been found yet. On the other hand, about 30% of the cells responded in stromal layers of long-term bone marrow cultures and prolonged CFU-GM production and cobblestone area formation were observed there, suggesting that cell-cell contact and adherence molecules play a regulatory role in PHSC replication.(ABSTRACT TRUNCATED AT 250 WORDS)

A protocol for the enrichment of different types of CFU-S from fetal mouse liver.

A method is described for purifying hemopoietic stem cells from fetal mouse liver. It entails three steps. First, fetal liver cell obtained from 14- and 15- days-old fetuses are centrifuged on a discontinuous metrizamide gradient and the low density fraction is removed. These cells are then incubated with monoclonal antibodies directed against late differentiation antigens, and the positive cells are removed by immunomagnetic beads. The negative cells are labelled with fluorescein-conjugated pokeweed mitogen (FITC-PWM) and sorted by a fluorescence- activated cell sorter (FACS) on the basis of differences in fluorescence intensity. The number of stem cells is determined by spleen colony assay (CFU-S) for the various sorted fractions. We observed that, as shown for the bone marrow, the different cell fractions are responsible for CFU-S heterogeneity in the kinetics of spleen colony formation. The PWM dull day-12 CFU-S, which originate from cells with a high enrichment factor, would probably be a closer measure of pluripotent hemopoietic stem cells (PHSC), while the PWM bright day-8 and day-12 CFU-S likely originate from committed stem cells. Furthermore, the PWM dull sorted cells had a better capacity for protecting lethally irradiated mice than did the bright cells, although the latter yielded good numbers of day-8 and day-12 CFU-S that displayed, as in the bone marrow, a discrepancy between the enrichment for day-12 CFU-S and radioprotection.