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.

Ex vivo expansion of hematopoietic stem cells for use in nonmyeloablative transplantation

“…Cell separation techniques were instrumental in leading to the discovery of a fraction of BM cells that was highly depleted of CFU-S but still capable of sustaining long-term hematopoiesis in vivo (Jones et al, 1989;Jones etal., 1990). Further studies using physically separated cell types confirmed that CFU-S have limited short-term repopulating capacity and are distinct from more primitive cells capable of multilineage repopulation for extended (> 2 months) period of times (Morrison and Weissman, 1994;Szilvassy et al, 1989;van der Loo et al, 1994) Definitive evidence of the existence of multipotent HSCs with lifelong lymphomyeloid repopulating activity came from clonal tracking studies. These were initially based on the use of radiation-induced chromosomal markers and later random genomic integration sites of retroviral vectors.…”

The Efficacy of Recombinant Thrombopoietin in Murine and Nonhuman Primate Models for Radiation‐Induced Myelosuppression and Stem Cell Transplantation

“…Recently, it was reported that transplantation of bone marrow from TPO-treated, 3.5 Gy-irradiated donor mice facilitated the 90-day survival of the recipient mice [86]. However, this survival effect had already become established at the short-term hematopoietic reconstitution parameter of 30-day survival (already in practice within 17 days), closely associated [82] to the CFU-S-13. Using such an experimental design to establish TPO effects on long-term repopulating cells would require a genetic marker able to distinguish between donor and recipient cells along multiple hemopoietic lineages.…”

1 Stem cells: characterization and measurement

“…Therefore, HSC assays commonly use these characteristics as an endpoint. In vivo, spleen colony formation (Till and McCulloch, 1961), animal survival, competitive repopulation (Harrison et al, 1988;Harrison 1992), retransplantation (Lemischka et al, 1986;Spangrude et al, 1995) and engraftment of mice having a congenital immune-suppression (Lapidot et al, 1992), anaemia (Boggs et al, 1982;Van der Loo et al, 1994;Ploemacher, 1994) or polymorphism in an enzyme (Down and Ploemacher, 1993) or cell surface epitope (Szilvassy et al, 1989;Pawliuk et al, 1996) have been used to detect HSCs and their descendants. In vitro, clonogenic potential (Pluznik and Sachs, 1965;Bradley and Metcalf, 1966) with particular attention for multipotential or blast-colony formation (Tsuji et al, 1991) and replating efficiency, shortterm liquid (delta) cultures (Muench and Moore, 1992;Moore and Hoskins, 1994;Petzer et al, 1996;Srour et al, 1996) and long-term progenitor cell production on stromal cells (Dexter et al, 1977;Gartner and Kaplan, 1980;Sutherland et al, 1990Sutherland et al, , 1991Breems et al, 1994) have been employed.…”

“…Extensive studies using physically sorted BMC have allowed regression analysis on the applicability of the murine CAFC assay as an in vitro equivalent for a series of HSC subsets in vivo. These studies have indicated that both in vivo and in vitro early developing, short-lived clones are initiated by the transient repopulating CFU-S cells (day-12), whereas later developing and more permanent clones are descendants of more primitive long-term repopulating HSCs, that induce stable chimerism for more than a year (Ploemacher et al, , 1993bPloemacher, 1994;Van der Loo et al, 1994). There is increasing evidence that the human CAFC assay may also detect transiently and long-term repopulating normal and leukaemic stem cell subsets (Winton and Colenda, 1987;Terpstra et al, 1996a,b;Cornelissen et al, 1997;Breems et al, 1996Breems et al, , 1997.…”

“…Analogous to the mouse, the human HSC compartment is likely to represent a hierarchy of primitive cells based on decreasing ability to generate new HSCs, decreasing pluripotentiality and proliferative potential, and increasing turnover rate (Rosendaal et al, 1979;Mauch et al, 1980;Hodgson and Bradley, 1984;Ploemacher and Brons, 1989;Ploemacher et al, , 1993bPloemacher, 1994). For transplants, this heterogeneity may be reflected in the different time periods which individual stem cell clones contribute to long-term reconstitution of a conditioned host, irrespective of whether this includes all haemopoietic lineages (Lemischka et al, 1986;Capel et al, 1990;Harrison, 1992;Lemischka, 1992;Van der Loo et al, 1994;Ploemacher, 1994). The question then arises as to what should be denoted as a 'stem cell'.…”