Rebecca H. Cho

A new mechanism for the aging of hematopoietic stem cells : aging changes the clonal composition of the stem cell compartment but not individual stem cells

Whether hematopoietic stem cells (HSCs) change with aging has been controversial. Previously, we showed that the HSC compartment in young mice consists of distinct subsets, each with predetermined self-renewal and differentiation behavior. Three classes of HSCs can be distinguished based on their differentiation programs: lymphoid biased, balanced, and myeloid biased. We now show that aging causes a marked shift in the representation of these HSC subsets. A clonal analysis of repopulating HSCs demonstrates that lymphoid-biased HSCs are lost and long-lived myeloid-biased HSCs accumulate in the aged. Myeloid-biased HSCs from young and aged sources behave similarly in all aspects tested. This indicates that aging does not change individual HSCs. Rather, aging changes the clonal composition of the HSC compartment. We show further that genetic factors contribute to the age-related changes of the HSC subsets. In comparison with B6 mice, aged D2 mice show a more pronounced shift toward myeloid-biased HSCs with a corresponding reduction in the number of both T- and B-cell precursors. This suggests that low levels of lymphocytes in the blood can be a marker for HSC aging. The loss of lymphoid-biased HSCs may contribute to the impaired immune response to infectious diseases and cancers in the aged.

Characterization and quantification of clonal heterogeneity among hematopoietic stem cells - a model-based approach

Hematopoietic stem cells (HSCs) show pronounced heterogeneity in self-renewal and differentiation behavior, which is reflected in their repopulation kinetics. Here, a single-cell-based mathematical model of HSC organization is used to examine the basis of HSC heterogeneity. Our modeling results, which are based on the analysis of limiting dilution competitive repopulation experiments in mice, demonstrate that small quantitative but clonally fixed differences of cellular properties are necessary and sufficient to account for the observed functional heterogeneity. The model predicts, and experimental data validate, that competitive pressures will amplify small clonal differences into large changes in the number of differentiated progeny. We further predict that the repertoire of HSC clones will evolve over time. Last, our results suggest that larger differences in cellular properties have to be assumed to account for genetically determined differences in HSC behavior as observed in different inbred mice strains. The model provides comprehensive systemic and quantitative insights into the clonal heterogeneity among HSCs with potential applications in predicting the behavior of malignant and/or genetically modified cells.

High frequency of long-term culture-initiating cells retain in vivo repopulation and self-renewal capacity

OBJECTIVE We wished to test if the long-term culture initiating cell (LTC-IC) assay measures primitive hematopoietic stem cells. An LTC-IC is defined by its ability to repopulate a stromal layer by forming colonies of myeloid cells. A negative well should never have received a stem cell, whereas a positive well should have been initiated by a stem cell. If these colonies were derived from stem cells, then a subset of the positive wells should retain stem cell activity. MATERIALS AND METHODS Limiting dilution cultures were initiated on the stromal cell line S17. Individual clonal cultures from LTC-IC assays were assessed for repopulation capacity in W(14)W(41) mice. RESULTS In long-term repopulation experiments, little activity was found in the negative wells, whereas 50% of the positive wells contained repopulating stem cells. The diverse in vivo repopulation patterns of the clonally derived stem cells suggest that this assay detects the full spectrum of stem cell types. Secondary transfers show that the clonally derived stem cells have self-renewal capacity. Experiments with mixtures of genetically distinguished cells showed that most (>90%) of the cultures were clonal. CONCLUSIONS Our data present the first formal link between LTC-IC and repopulating stem cells. Moreover, the culture system presents a new way of generating a high frequency of clonally repopulating stem cells.

Limiting dilution analysis for estimating the frequency of hematopoietic stem cells: Uncertainty and significance

The ability to predict accurately the number of hematopoietic stem cells (HSCs) in a graft is important for the success of HSC transplantation. Limiting dilution analysis (LDA) in vitro and in vivo is widely used to enumerate HSCs. However, there have been few attempts to standardize this approach. Particularly, the role of statistical and experimental errors in the performance and evaluation of LDA has received little attention. Since these errors directly affect the interpretation, validity, and significance of the LDA results, we have here performed a systematic analysis of the contribution of different types of errors.Long-term culture-initiating cells (LTC-IC) in the bone marrow of C57BL/6 (B6) mice were measured. Experiments were designed to exclude systematically different types of experimental errors. Computer simulations were performed to estimate the statistical error. Analysis of 137 LTC-IC assays showed 2.8 +/- 1.06 LTC-IC per 10(5) cells in the bone marrow of B6 mice. The major components of the uncertainty were derived from variations introduced by performing the experiments at different time points and by the statistical error. Surprisingly, operator errors and mouse-to-mouse error, including age and sex of the animals, contributed little to the overall uncertainty. As expected, the errors were found to decrease when increasing numbers of replica were analyzed. A computer program was developed to assist with the optimal design of the assay. The analysis presented here provides rational strategies for standardizing the experimental design and for gauging the accuracy of LDA-based HSC measurements.