G de Haan

A putative gene causes variability in lifespan among genotypically identical mice

Ageing is a complex process during which damage to multiple organ systems accumulates1. The time at which impairment of critical bodily functions becomes incompatible with life determines organismal longevity and is usually measured in a population as mean or maximal lifespan. Our current understanding of the genes affecting longevity, particularly in mammalian species, is rudimentary at best2. Here we studied the range of lifespans among individual animals within genetically identical recombinant inbred (RI) strains of mice. We found that the time between the deaths of the first and last members of a given strain fell into two temporal groups, despite the fact that strains may have had similar mean or maximal lifespans. The segregation of RI strains into wide and narrow ranges that roughly corresponded to characteristic lifespan ranges of the progenitor strains, suggested a genetic component. We therefore mapped a putative gene accounting for more than 60% of the phenotype to a genomic segment near the centromere on chromosome 11. This locus causes variability in the rate at which genetically identical members of a population die and, from a practical standpoint, affects the slope of a death curve for a population.

Conditioning regimens using growth factors prior to low dose tbi enhance engraftment of murine stem cells after bone marrow transplantations

Abstract Low toxic regimens to prepare recipients of autologous and allogeneic bone marrow transplantations (BMT) are currently being explored extensively for an expanding variety of clinical applications. We have developed a novel conditioning protocol with the aim to enhance donor cell engraftment after low dose total body irradiation (TBI). Autologous BMT was performed in mice pretreated with early acting growth factors (GF) prior to irradiation to increase the radiosensitivity of host bone marrow cells, which should lead to their selective depletion. Mice (C57BL/6, Ly5.2) were treated 1, 3, or 7 days with SCF (2.5 μg/day) and IL-11 (2.0 μg/day) before sublethal (4Gy) TBI. Survival of radioresistant subsets of host stem cells was assessed using the CAFC assay. We analyzed % donor chimerism post-transplantation after infusion of 3×10 6 Ly5.1 BM cells by FACS analysis. The CAFC data accurately predicted both short- and long-term development of donor cell engraftment. One-day pretreated mice showed a 2-fold increase in remaining host CAFC subsets after TBI compared to untreated, irradiated mice. The elevated host CAFC subsets were associated with low donor leukocyte chimerism (

Autonomous regulation of stem cell frequency

Abstract Lineage restricted growth factors regulate peripheral blood cell homeostasis. However, mechanisms that affect the frequency of primitive stem cells with long term proliferative potential remain largely unknown. We have shown that stem cell numbers in DBA/2 (D2) mice are higher than in C57BL/6 (B6) mice. To assess whether this is a cell-extrinsic (e.g. due to variation of growth factor concentrations) or cell-autonomous trait we have developed a model in which D2 and B6 stem cells co-exist is a single animal. Chimeric mice were produced by transplanting irradiated B6D2F1 recipients with mixtures of D2 and B6 day-14 fetal liver cells. Donor leukocyte contribution was assessed using anti-H2K b/d antibodies. In agreement with our previous findings, repopulating ability of D2 fetal liver cells was ∼10 fold higher than B6 cells. In order to determine the actual frequency of D2 and B6 stem and progenitor cells in these chimeric animals, we performed several experiments. Firstly, mice in which D2 and B6 leukocyte contributions were equal, were injected with G-CSF. Peripheral blood CFU-GM were cultured and the genotype of each colony was assessed using SSLP-PCR. D2 CFU-GM were 4-8 fold more numerous than B6 progenitors. Marrow was harvested from these chimeric mice and D2 and B6 cell populations were separated by flow cytometry. CAFC analysis on sorted samples showed that the frequency of D2 late appearing CAFC subsets was ∼10 fold higher, indicating that chimerism of mobilized progenitors reflected marrow stem cell chimerism. To confirm these in vitro data we performed a secondary transplantation using unfractionated chimeric marrow, transplanted in limiting doses to F1 recipients. Comparison of the proportion of animals showing D2 and B6 leukocyte contributions revealed that the frequency of D2 LTRA was ∼ 10 fold higher than B6 LTRA numbers. Our data demonstrate that two genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotype/frequency. This indicates that stem cells maintain their population size autonomously.