Christa E. Müller-Sieburg

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

OBJECTIVEnWe 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.nnnMATERIALS AND METHODSnLimiting 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.nnnRESULTSnIn 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.nnnCONCLUSIONSnOur 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.

Stem cell aging: survival of the laziest?

The question whether stem cells age remains an enigma. Traditionally, aging was thought to change the properties of hematopoietic stem cell (HSC). We discuss here a new model of stem cell aging that challenges this view. It is now well-established that the HSC compartment is heterogeneous, consisting of epigenetically fixed subpopulations of HSC that differ in self-renewal and differentiation capacity. New data show that the representation of these HSC subsets changes during ageing. HSC that generate lymphocyte-rich progeny are depleted, while myeloid-biased HSC are enriched in the aged HSC compartment. Myeloid-biased HSC, even when isolated from young donors, have most of the characteristics that had been attributed to aged HSC. Thus, the distinct behavior of the HSC isolated from aged hosts is due to the accumulation of myeloid-biased HSC. By extension this means that the properties of individual HSC are not substantially changed during the lifespan of the organism and that aged hosts do not contain many aged HSC. Myeloid-biased HSC give rise to mature cells slowly but contribute for a long time to peripheral hematopoiesis. We propose that such slow, “lazy” HSC are less likely to be transformed and therefore may safely sustain hematopoiesis for a long time.

Clonal diversity of the stem cell compartment.

Purpose of reviewHematopoietic stem cells are functionally heterogeneous even when isolated as phenotypically homogenous populations. How this heterogeneity is generated is incompletely understood. Several models have been formulated to explain the generation of diversity. All of these assume the existence of a single type of hematopoietic stem cell that generates heterogeneous daughter stem cells in response to extrinsic or intrinsic (stochastic) signals. This view has encouraged the idea that stem cells can be instructed to adapt their function. Newer data, however, challenge this concept. Here, we summarize these findings and discuss their implication for applications of stem cells. Recent findingsHematopoietic stem cells that differ in function have been documented during development and within the adult stem cell compartment. The differences in function are stably inherited to daughter stem cells when these cells proliferate to self-renew. Collectively, the data show that the adult stem cell compartment consists of a limited number of distinct classes of stem cells. SummaryThe most important stem cell functions, including self-renewal and differentiation capacity, are preprogrammed through epigenetic or genetic mechanisms. Thus, stem cells are much more predictable than previously thought. Changes in the stem cell compartment through disease or aging can be interpreted as shifts in its clonal composition, rather than a modification of individual hematopoietic stem cells.

Predicting clonal self-renewal and extinction of hematopoietic stem cells

A single hematopoietic stem cell (HSC) can generate a clone, consisting of daughter HSCs and differentiated progeny, which can sustain the hematopoietic system of multiple hosts for a long time. At the same time, this massive expansion potential must be restrained to prevent abnormal, leukemic proliferation. We used an interdisciplinary approach, combining transplantation assays with mathematical and computational methods, to systematically analyze the proliferative potential of individual HSCs. We show that all HSC clones examined have an intrinsically limited life span. Daughter HSCs within a clone behaved synchronously in transplantation assays and eventually exhausted at the same time. These results indicate that each HSC is programmed to have a finite life span. This program and the memory of the life span of the mother HSC are inherited by all daughter HSCs. In contrast, there was extensive heterogeneity in life spans between individual HSC clones, ranging from 10 to almost 60 mo. We used model-based machine learning to develop a mathematical model that efficiently predicts the life spans of individual HSC clones on the basis of a few initial measurements of donor type cells in blood. Computer simulations predict that the probability of self-renewal decays with a logistic kinetic over the life span of a normal HSC clone. Other decay functions lead to either graft failure or leukemic proliferation. We propose that dynamical fate probabilities are a crucial condition that leads to self-limiting clonal proliferation.

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.

Lifespan Differences in Hematopoietic Stem Cells are Due to Imperfect Repair and Unstable Mean-Reversion

The life-long supply of blood cells depends on the long-term function of hematopoietic stem cells (HSCs). HSCs are functionally defined by their multi-potency and self-renewal capacity. Because of their self-renewal capacity, HSCs were thought to have indefinite lifespans. However, there is increasing evidence that genetically identical HSCs differ in lifespan and that the lifespan of a HSC is predetermined and HSC-intrinsic. Lifespan is here defined as the time a HSC gives rise to all mature blood cells. This raises the intriguing question: what controls the lifespan of HSCs within the same animal, exposed to the same environment? We present here a new model based on reliability theory to account for the diversity of lifespans of HSCs. Using clonal repopulation experiments and computational-mathematical modeling, we tested how small-scale, molecular level, failures are dissipated at the HSC population level. We found that the best fit of the experimental data is provided by a model, where the repopulation failure kinetics of each HSC are largely anti-persistent, or mean-reverting, processes. Thus, failure rates repeatedly increase during population-wide division events and are counteracted and decreased by repair processes. In the long-run, a crossover from anti-persistent to persistent behavior occurs. The cross-over is due to a slow increase in the mean failure rate of self-renewal and leads to rapid clonal extinction. This suggests that the repair capacity of HSCs is self-limiting. Furthermore, we show that the lifespan of each HSC depends on the amplitudes and frequencies of fluctuations in the failure rate kinetics. Shorter and longer lived HSCs differ significantly in their pre-programmed ability to dissipate perturbations. A likely interpretation of these findings is that the lifespan of HSCs is determined by preprogrammed differences in repair capacity.