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.
Corso
A
A
Divisione di Ematologia, Policlinico San Matteo, Pavia, Italy.
Hogeweg-Platenburg
M G
MG
De Vries
P
P
Visser
J W
JW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001854:Bone Marrow Cells; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003114:Colony-Forming Units Assay; D005260:Female; D016332:Fetal Tissue Transplantation; D005434:Flow Cytometry; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells; D007163:Immunosorbent Techniques; D008099:Liver / Q000166:cytology* / Q000196:embryology; D008280:Magnetics; D008297:Male; D051379:Mice / Q000196:embryology; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D011043:Pokeweed Mitogens; D011828:Radiation Chimera
D000911:Antibodies, Monoclonal; D011043:Pokeweed Mitogens