Stable multilineage hematopoietic chimerism in alpha-thalassemic mice induced by a bone marrow subpopulation that excludes the majority of day-12 spleen colony-forming units

Abstract

We have investigated the contribution of highly purified day-12 spleen colony-forming units (CFU-S-12) as well as more primitive cells to sustained blood cell production using in vivo and in vitro assays that allow frequency analysis. Normal or day-6 post-5-fluorouracil light- density bone marrow (BM) was sorted on the basis of differences in rhodamine-123 (Rh123) retention or wheat germ agglutinin (WGA) affinity and tested in vivo using a recently developed alpha-thalassemic chimeric mouse model. In addition, short-term and long-term clonal activity was assessed in vitro using a limiting dilution-type long-term BM culture, the cobblestone area forming cell assay. When sublethally irradiated alpha-thalassemic mice were transplanted with as many as 281 purified WGAbright CFU-S-12, derived from a fraction containing 95% of all CFU-S-12 from day-6 post-5-fluorouracil light-density BM of wild- type mice, detectable chimerism was not observed at 6 months posttransplantation. In contrast, only three CFU-S-12 were included in the Rh123dull and WGAdim subpopulations that induced 29% to 58% and 21% to 31% stable multilineage donor-type chimerism of erythrocytes and leukocytes, respectively. The Rh123dull and WGAdim cells were up to 240- fold enriched for long-term repopulating ability (LTRA) as compared with unseparated BM. A comparable level of chimerism was found in the different hematopoietic organs and at the level of BM CFU-S-12. The frequency of the LTRA unit capable of inducing a 10% sustained level of donor-type erythrocytes was calculated to be 1 to 2 per 10(5) BM cells. Several reports have suggested that LTRA and spleen colony formation could be capacities of the same stem cell subset. However, the present results show that the majority of CFU-S-12 have only short-term repopulating ability and are physically separable from more primitive stem cells with long-term multilineage reconstituting capacities.

Separation of G‐CSF‐mobilized PBSC transplants by counterflow centrifugal elutriation: modest enrichment of CD34+ cells but no loss of primitive haemopoietic progenitors

“…The frequencies of CAFC clones of normal appearance observed in cultures inoculated with cells from lymphocyteenriched CCE fractions decreased during the course of the cultures. Week type 6 CAFC, which, as in the well-validated murine CAFC-assay, may be considered as indicators of stem cells capable of long-term haemopoietic reconstitution (Breems et al, 1994(Breems et al, , 1996Murray et al, 1995;Ploemacher et al, 1993;Van der Loo et al, 1994;Ploemacher, 1994), were nearly exclusively recovered in the elutriation fractions containing the large cells. These results indicate that primitive G-CSF mobilized stem cells were not separated from colony-forming units in the CCE procedure used in this study.…”