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During aging, a decline in stem cell function is observed in many tissues. This decline is accompanied by complex changes of the chromatin structure among them changes in histone modifications and DNA methylation which both affect transcription of a tissue-specific subset of genes. A mechanistic understanding of these age-associated processes, their interrelations and environmental dependence is currently lacking. Here, we discuss related questions on the molecular, cellular, and population level. We combine an individual cell-based model of stem cell populations with a model of epigenetic regulation of transcription. The novel model enables to simulate age-related changes of trimethylation of lysine 4 at histone H3 and of DNA methylation. These changes entail expression changes of genes that induce age-related phenotypes (ARPs) of cells. We compare age-related changes of regulatory states in quiescent stem cells occupying a niche with those observed in proliferating cells. Moreover, we analyze the impact of the activity of the involved epigenetic modifiers on these changes. We find that epigenetic aging strongly affects stem cell heterogeneity and that homing at stem cell niches retards epigenetic aging. Our model provides a mechanistic explanation how increased stem cell proliferation can lead to progeroid phenotypes. Adapting our model to properties observed for aged hematopoietic stem cell (HSC) clones, we predict that the hematopoietic ARP activates young HSCs and thereby retards aging of the entire HSC population. In addition, our model suggests that the experimentally observed high interindividual variance in HSC numbers originates in a variance of histone methyltransferase activity., (© 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Background: Hematopoiesis is a complex process involving different cell types and feedback mechanisms mediated by cytokines. This complexity stimulated various models with different scopes and applications. A combination of complementary models promises to provide their mutual confirmation and to explain a broader range of scenarios. Here we propose a combination of an ordinary differential equation (ODE) model of human granulopoiesis and an agent-based model (ABM) of hematopoietic stem cell (HSC) organization. The first describes the dynamics of bone marrow cell stages and circulating cells under various perturbations such as G-CSF treatment or chemotherapy. In contrast to the ODE model describing cell numbers, our ABM focuses on the organization of individual cells in the stem population., Results: We combined the two models by replacing the HSC compartment of the ODE model by a difference equation formulation of the ABM. In this hybrid model, regulatory mechanisms and parameters of the original models were kept unchanged except for a few specific improvements: (i) Effect of chemotherapy was restricted to proliferating HSC and (ii) HSC regulation in the ODE model was replaced by the intrinsic regulation of the ABM. Model simulations of bleeding, chronic irradiation and stem cell transplantation revealed that the dynamics of hybrid and ODE model differ markedly in scenarios with stem cell damage. Despite these differences in response to stem cell damage, both models explain clinical data of leukocyte dynamics under four chemotherapy regimens., Conclusions: ABM and ODE model proved to be compatible and were combined without altering the structure of both models. The new hybrid model introduces model improvements by considering the proliferative state of stem cells and enabling a cell cycle-dependent effect of chemotherapy. We demonstrated that it is able to explain and predict granulopoietic dynamics for a large variety of scenarios such as irradiation, bone marrow transplantation, chemotherapy and growth factor applications. Therefore, it promises to serve as a valuable tool for studies in a broader range of clinical applications, in particular where stem cell activation and proliferation are involved.

The kinetics of label uptake and dilution in dividing stem cells, e.g., using Bromodeoxyuridine (BrdU) as a labeling substance, are a common way to assess the cellular turnover of all hematopoietic stem cells (HSCs) in vivo. The assumption that HSCs form a homogeneous population of cells which regularly undergo cell division has recently been challenged by new experimental results. For a consistent functional explanation of heterogeneity among HSCs, we propose a concept in which stem cells flexibly and reversibly adapt their cycling state according to systemic needs. Applying a mathematical model analysis, we demonstrate that different experimentally observed label dilution kinetics are consistently explained by the proposed model. The dynamically stabilized equilibrium between quiescent and activated cells leads to a biphasic label dilution kinetic in which an initial and pronounced decline of label retaining cells is attributed to faster turnover of activated cells, whereas a secondary, decelerated decline results from the slow turnover of quiescent cells. These results, which support our previous model prediction of a reversible activation/deactivation of HSCs, are also consistent with recent findings that use GFP-conjugated histones as a label instead of BrdU. Based on our findings we interpret HSC organization as an adaptive and regulated process in which the slow activation of quiescent cells and their possible return into quiescence after division are sufficient to explain the simultaneous occurrence of self-renewal and differentiation. Furthermore, we suggest an experimental strategy which is suited to demonstrate that the repopulation ability among the population of label retaining cells changes during the course of dilution.

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.

Lineage specification of hematopoietic stem cells is considered a progressive restriction in lineage potential. This view is consistent with observations that differentiation and lineage specification is preceded by a low-level coexpression of lineage specific, potentially antagonistic genes in early progenitor cells. This coexistence, commonly referred to as priming, disappears in the course of differentiation when certain lineage-restricted genes are upregulated while others are downregulated. Based on this phenomenological description, we propose a quantitative model that describes lineage specification as a competition process between different interacting lineage propensities. The competition is governed by environmental stimuli promoting a drift from a multipotent coexpression to the dominance of one lineage. The assumption of a context-dependent intracellular differentiation control is consistently embedded into our previously proposed model of hematopoietic stem cell organization. The extended model, which comprises self-renewal and lineage specification, is verified using available data on the lineage specification potential of primary hematopoietic stem cells and on the differentiation kinetics of the FDCP-mix cell line. The model provides a number of experimentally testable predictions. From our results, we conclude that lineage specification is best described as a flexible and temporally extended process in which lineage commitment emerges as the result of a sequence of small decision steps. The proposed model provides a novel systems biological view on the functioning of lineage specification in adult tissue stem cells and its connections to the self-renewal properties of these cells. Disclosure of potential conflicts of interest is found at the end of this article.

Objective: At present, no dynamic quantitative models of stem cell organization are available that fulfill all criteria of the prevalent functional definition of hematopoietic stem cells and, at the same time, provide a consistent explanation of cell kinetic and functional stem cell heterogeneity, reversibility of cellular properties, self-organized regeneration after damage, fluctuating activity and competition of stem cell clones, and microenvironment dependency of stem cell quality. To solve this problem, we propose a new, comprehensive model concept., Materials and Methods: A single cell-based stochastic model is described. It makes the novel concept of within-tissue plasticity operational. Within a range of potential options, individual cells may reversibly change their actual set of properties depending on the influence of the local growth environment. Stochastic switching between the growth environments introduces fluctuations that eventually generate heterogeneity. Extensive model simulations are compared with experimental data., Results: Although stemness is not an explicit cellular model property, the system behavior is consistent with the functional definition of stem cells and explains a large set of experimental observations on stem cell function in vivo and in vitro on the level of cell populations and individual cells. Classic results such as the colony-forming unit spleen assay, as well as recent experimental observations on stem cell kinetics, individual clone tracking, and fluctuating clonal contribution, are discussed., Conclusions: This concept introduces a fundamentally new perspective on stem cell organization treating stemness not as an explicit cellular property but as the result of a dynamic process of self-organization. The model needs to be extended to incorporate lineage specification and tissue plasticity.

The aim of this study was to expand the primitive and committed hematopoietic cell compartments in vivo in order to confer resistance of the blood cell forming system against the cytotoxic, cell cycle specific drug 5-fluorouracil (5-FU). Possible chemoprotective effects of such a pretreatment could result from increased numbers of hematopoietic cells, present before 5-FU administration. In addition, we hypothesized that an enhanced number of primitive and progenitor calls would result in a reduced cycling activity, ie, 5-FU sensitivity, of these same cells, due to normal physiological feedback loops. Administration of stem cell factor (SCF) plus interleukin-11 (IL-11) to mice was shown to result in expansion of the various immature cell compartments in marrow and, in particular, spleen. The total body content of the primitive cobblestone area forming cells (CAFC)-day 28 was increased to 140%, whereas the more committed cells (CAFC-day 7, erythroid and granuloid progenitors) were increased to 500%. This in vivo expansion resulted in a decreased 5-FU sensitivity of the hematopoietic system. In particular, mice that had received 5-FU 24 hours after discontinuation of growth factor pretreatment showed significantly less toxicity of committed cell stages. Compared with mice not pretreated, it appeared that in pretreated mice, 24 hours after 5-FU administration, the absolute number, but also the fraction of surviving CAFC, was much higher in both marrow and spleen. This was caused by a decrease in the cycling activity of all primitive cell subsets. To explore the possible use of this finding in a chemotherapeutic setting, we determined the interval between two subsequent doses of 5-FU (160 mg/kg) that was required to prevent drug-induced mortality. When control mice received a second dose of 5-FU 7, 10, or 14 days after the first, respectively 0%, 20%, and 80% survived. In contrast, 40% and 100% of mice that received SCF + IL-11 before the first dose of 5-FU, survived a second dose of 5-FU given respectively after 7 or 10 days. To assess whether chemoprotection in this setting could be ascribed to protection of the hematopoietic system, we transplanted a high number of normal bone marrow cells (sufficient to compensate for any hematopoietic deficiency) to normal and pretreated mice after they had been administered 2 doses of 5-FU, given 7 days apart. Bone marrow transplantation (BMT) could only rescue 50% of mice not pretreated, showing that a significant part of the mortality was because of nonhematologic toxicity. However, a BMT given to growth factor pretreated mice saved all mice, indicating that in this setting SCF + IL-11 had additional protective effects on cell systems other than hematopoiesis. In conclusion, our study showed fundamental knowledge about the behavior of primitive cells in vivo and has shown that manipulation of these and other cell compartments with appropriate growth factors may confer resistance against cytotoxic drugs.

Agents with stem cell-toxic potential are frequently used for salvage therapy of Hodgkin's disease (HD) and high-grade non-Hodgkin's lymphoma (NHL). Because many patients with relapsed or refractory lymphoma are candidates for autologous progenitor cell transplantation, possible toxic effects of salvage chemotherapy on progenitor cells must be taken into account. In a retrospective study, we have analyzed the influence of a salvage regimen containing the stem cell-toxic drugs BCNU and melphalan (Dexa-BEAM) on subsequently harvested bone marrow (BM)- and peripheral blood-derived progenitor cell grafts (PBPC) and compared it with other factors. Progenitor cells were collected from 96 patients with HD or high-grade NHL. Seventy-nine grafts were reinfused (35 PBPC and 44 BM) after high-dose chemotherapy. Compared with patients autografted with BM, hematopoietic recovery was significantly accelerated in recipients of PBPC. For PBPC, the number of Dexa-BEAM cycles ( > or = v > 1) was the predominate prognostic factor affecting colony-forming unit-granulocyte-macrophage (CFU-GM) yield (66 v 6.8 x 10(4)/kg, P = .0001), CD34+ cell yield (6.6 v 1.6 x 10(6)/kg, P = .0001), neutrophil recovery to > 0.5 x 10(9)/L (9 v. 11 days, P = .0086), platelet recovery to > 20 x 10(9)/L (10 v 15.5 days, P = .0002), and platelet count on day +100 after transplantation (190 v 107 x 10(9)/L, P = .031) using univariate analysis. Previous radiotherapy was associated with significantly lower CFU-GM and CD34+ cell yields but had no influence on engraftment. Patient age, patient sex, disease activity, or chemotherapy other than Dexa-BEAM did not have any prognostic impact. Multivariate analysis confirmed that Dexa-BEAM chemotherapy was the overriding factor adversely influencing CFU-GM yield (P < .0001), CD34+ cell yield (P < .0001), and platelet engraftment (P < .0001). BM grafts were not significantly affected by previous Dexa-BEAM chemotherapy or any other variable tested. However, prognostic factors favoring the use of BM instead of PBPC were not identified using joint regression models involving interaction terms between the graft type (PBPC or BM) and the explanatory variables investigated. We conclude that, in contrast to previous radiotherapy or other chemotherapy, exposure to salvage regimens containing stem cell-toxic drugs, such as BCNU and melphalan, is a critical factor adversely affecting yields and performance of PBPC grafts. Marrow progenitor cells appear to be less sensitive to stem cell-toxic chemotherapy. PBPC should be harvested before repeated courses of salvage chemotherapy involving stem cell-toxic drugs to preserve the favorable repopulation kinetics of PBPC in comparison with BM.

Because of the complexity of appropriate stem cell assays, little information on the in vivo regulation of murine stem cell biology or stemmatopoiesis is available. It is unknown whether and how in vivo the primitive hematopoietic stem cell compartment is affected during a continued increased production of mature blood cells. In this study, we present data showing that prolonged (3 weeks) administration of granulocyte colony-stimulating factor (G-CSF), which is a major regulator of mature granulocyte production, has a substantial impact on both the size and the location of various stem cell subset pools in mice. We have used the novel cobblestone area forming cell (CAFC) assay to assess the effects of G-CSF on the stem cell compartment (CAFC days 7, 14, 21, and 28). In marrow, in which normally 99% of the total number of stem cells can be found, G-CSF induced a severe depletion of particularly the most primitive stem cells to 5% to 10% of normal values. The response after 7 days of G-CSF treatment was an increased amplification between CAFC day 14 and 7. However, this response occurred at the expense of the number of CAFC day 14. It is likely that the resulting gap of CAFC day 14 cell numbers was subsequently replenished from the more primitive CAFC day 21 and 28 compartments, because these cell numbers remained low during the entire treatment period. In the spleen, the number of stem cells increased, likely caused by a migration from the marrow via the blood, leading to an accumulation in the spleen. The increased number of stem cells in the spleen overcompensated for the loss in the marrow. When total body (marrow and spleen) stem cell numbers were calculated, it appeared that a continued increased production of mature granulocytes resulted in the establishment of a higher, new steady state of the stem cell compartment; most committed stem cells (CAFC day 7) were increased threefold, CAFC day 14 were increased 2.3-fold, CAFC-day 21 were increased 1.8-fold, and the most primitive stem cells evaluated, CAFC day 28, were not different from normal, although now 95% of these cells were located in the spleen. Four weeks after discontinuation of the G-CSF treatment, the stem cell reserve in the spleen had returned to a normal level, whereas stem cell numbers in marrow had recovered to values above normal. This study shows that the primitive stem cell compartment is seriously perturbed during an increased stimulation of the production of mature blood cells.(ABSTRACT TRUNCATED AT 400 WORDS)

In this study, we determined in vivo interactions between hemopoietic growth factors and etoposide (VP-16) to assess whether normal blood cell production could be maintained during chemotherapy if hemopoietic growth factors were simultaneously administered. Groups of mice were treated for 7 consecutive days with four different doses of VP-16 in combination with three different doses of erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF). In total, 12 combinations of VP-16 plus EPO and 12 combinations of VP-16 plus G-CSF were thus evaluated. Intricate dose-response surfaces of the effects of the different treatments on colony-forming units-erythroid, reticulocytes, hematocrit, colony-forming units-granulocyte/macrophage, and absolute neutrophil count were obtained, which revealed that: (a) simultaneous EPO administration was able to maintain reticulocyte production and to protect mice from VP-16 induced anemia; (b) simultaneous G-CSF administration was able to maintain granulocyte production and to protect mice from VP-16 induced neutropenia; (c) VP-16 dose escalation was feasible when EPO or G-CSF were simultaneously administered; and (d) no increased myelotoxicity on erythroid or granuloid progenitors was observed when EPO or G-CSF was simultaneously administered with VP-16. These results suggest that in vivo either individual hemopoietic progenitors can become resistant against VP-16-induced cell death by appropriate simultaneous growth factor administration or that the loss of overall cell amplification, induced by VP-16, can be compensated by extra amplification of surviving progenitors. Furthermore, these data indicate that a strict separation in time of cytostatic drug and growth factor treatment is not necessarily the optimal schedule with respect to the reduction of hemotoxicity.

The hematopoietic cell response to benzene intoxication in mice (during and after long-term inhalation) was analyzed by a mathematical model of murine hematopoiesis. Two complementary methods, Time-Curve and Steady-State Analysis, were developed to identify target cells for benzene toxicity and to quantify the extent of damage in different stages of development of these target cells. We found that (i) erythropoietic cells were the most sensitive; (ii) granulopoietic cells were about half as sensitive as erythropoietic and (iii) hematopoietic stem cells exhibited a sensitivity that ranged between that of erythropoietic and granulopoietic cells. A dose-response relationship between benzene levels and damage in target cells (valid from 1 to more than 900 ppm) was derived that was linear for doses up to 300 ppm and plateaued thereafter. This relationship indicated that benzene-induced hematotoxicity is subject to a saturable process. Recovery of hematopoiesis following chronic benzene intoxication was simulated for different doses and preceding exposure periods. The impaired recovery following exposure periods greater than 8 weeks could be explained by a severe reduction in the maximum self-maintenance of stem cells. This study indicates that the present mathematical model represents a useful approach to investigate alternate hypotheses for the action of hematotoxic agents.

Previously it has been hypothesized that the granulopoietic and erythropoietic lineages may compete for differentiating stem cells. According to this hypothesis one would expect that a stimulation of granulopoiesis by G-CSF administration would lead to a reduction of the stem cell pool and be followed by a decline of erythropoietic progenitor numbers. In addition one would expect an enhanced response of granulopoiesis if G-CSF administration were combined with suppression of erythropoiesis by red cell transfusion. To evaluate whether this hypothesis holds true C57bl mice were injected subcutaneously for 6 d with 3.75 micrograms rh G-CSF/mouse/d (150 micrograms G-CSF/kg body weight/d). Marrow CFU-S numbers showed an increase to 160% on day 2, followed by a decrease to 50% of control on day 6. Splenic and peripheral blood CFU-S increased 20-fold and 10-fold, respectively. Marrow CFU-E declined to 40% of the control value. Splenic CFU-E increased 10-fold. The increase in marrow CFU-GM numbers ranged between 140% and 180%. CFU-GM obtained from the spleen and the peripheral blood increased 60-fold and 15-fold, respectively. Regarding the CFU-S and CFU-GM a similar pattern of response was found in an experiment where rh G-CSF administration was combined with an additional red cell transfusion. These data do not provide convincing evidence for an exhaustion of haemopoietic stem cells during treatment with G-CSF. They rather suggest that an important side effect of G-CSF treatment is a release of CFU-S and progenitors from the marrow to the peripheral blood and a reseeding in the spleen.

Recovery of hemopoiesis was studied after a 3-day treatment with the antibiotic thiamphenicol (TAP). A contrasting behavior of the spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and erythroid colony-forming units (CFU-E) numbers in the bone marrow versus those in the spleen was found. Whereas the cell numbers reached nadirs in the marrow, they peaked 30 to 100-fold above control values in the spleen on day 4. Simultaneously the number of CFU-S, BFU-E, and CFU-GM, but not of CFU-E, increased drastically in the peripheral blood. The tritiated thymidine kill of the splenic CFU-S was too small to explain the endogenous splenic production of these cells. A quantitative analysis further revealed that an effective erythropoiesis was established in the spleen. As a consequence, the first part of a reticulocytosis was mainly due to the splenic contribution, whereas the second part predominantly originated from a delayed marrow erythropoiesis. In contrast, the CFU-GM of the spleen did not effectively differentiate into granuloid precursors. The bulk of the granuloid production occurred in the marrow. The best explanation for these results is a net migration of CFU-S, BFU-E, and CFU-GM from the marrow to the spleen during early recovery, and a back-migration of CFU-GM to the marrow later in the recovery phase. These observations indicate a link between migration of hemopoietic cells and their differentiation at the two hemopoietic sites.

On the basis of experimental knowledge about haemopoietic stem cells a catalogue of fundamental statements is formulated. From this a simple mathematical model of haemopoietic regulation mechanisms is developed. The functional net effects of regulatory processes which are still unknown or unmeasurable are estimated using evolution arguments. The model is developed in three steps. It allows description of the self-replication of stem cells after direct destruction as well as their reaction to increased or reduced needs in the erythropoietic system. The most important experimental data about changes in CFUs, BFUe and CFUe after acute or chronic irradiation, anaemia, hypoxia, hypertransfusion or direct erythropoietic stimulation can be reproduced within the model. The model allows us to understand most of the results of experimental stem cell research. Furthermore, it can be applied for a more precise analysis of the existing data. Predictions about the results of certain experiments can be made.

A mathematical model has been developed which describes the regulation of erythropoiesis in mice and rats. The main model assumptions are: (1) Regulation is mediated by erythropoietin (EPO). (2) The production of EPO depends exponentially on the tissue oxygen pressure (e.g. in the renal production sites). (3) There are sigmoidal dose-response curves relating the EPO concentration in the plasma to the mitotic activity of CFU-E and proliferative erythropoietic precursors. For maximum stimulation two to four additional mitoses may occur, while for an absent stimulus three to five mitoses may be omitted. (4) The normal precursor transit time of three to four days may be shortened by more than 50% during maximum stimulation. (5) The erythrocytes have a normal lifespan of 42-56 days, which may be reduced to 15-20 days under erythropoietic stimulation. Among these assumptions, the dose-response relationships between EPO and the mitotic activity of CFU-E and the proliferative erythropoietic precursors are the most important hypotheses of the model. This is the first of a series of three papers and gives a description of the mathematical formalism and the parameters used. In the subsequent papers computer simulations on erythropoietic stimulation and suppression are presented.

A previously described mathematical model of the hematopoietic stem cell system has been extended to permit a detailed understanding of the data during and after hypoxia. The model includes stem cells, erythroid and granuloid progenitors and precursors. Concerning the intramedullary feedback mechanisms two basic assumptions are made: 1) The fraction "a" of CFU-S in active cell cycle is regulated. Reduced cell densities of CFU-S, progenitors or precursors lead to an accelerated stem cell cycling. Enlarged cell densities suppress cycling. 2) The self renewal probability "p" of CFU-S is also regulated. The normal steady state is described by p = 0.5, indicating that on statistical average each dividing mother stem cell is replaced by one daughter stem cell, while the second differentiates. Diminished cell densities of CFU-S or enlarged densities of progenitors and precursors induce a more intensive self renewal (p greater than 0.5), such that the stem cell number increases. The self renewal probability declines (p less than 0.5) if too many CFU-S or too few progenitors and precursors are present. The model reproduces bone marrow data for CFU-S, BFU-E, CFU-C, CFU-E, 59 Fe-uptake and nucleated cells in hypoxia and posthypoxia. Although the ratio of differentiation into the erythroid and granuloid cell lines is kept constant in the model, a changing ratio of CFU-E and CFU-C results. The model suggests that stem cells and progenitor cells are regulated by a regulatory interference of erythropoiesis and granulopoiesis.

Supplementary to the discussion in Blood Cells 7 (1981) pp. 417-443 concerning regulatory mechanisms of stem cell proliferation and differentiation, the consequences of the two proposed hypotheses of Blackett and Botnick and Lord have been calculated. It can be shown that their equations are equivalent and can be transformed into each other. However, their verbally described regulatory mechanisms are different and turn out to be more important than the equations. The assumptions of Blackett and Botnick lead to an unstable behaviour of the stem cell system with cyclic cell numbers while the hypothesis of Lord leads to stable recovery curves.

Continuous treatment of C57bl/6 mice for 4 days with the cytostatic antibiotic thiamphenicol revealed a dual response of hemopoietic cells. On one hand, morphologically recognizable erythroid precursors and late progenitors (erythroid colony-forming units; CFU-E) and, to a lesser extent, granuloid precursors were found substantially reduced. On the other hand, early granuloid (granulocyte-macrophage colony-forming units; CFU-GM) and erythroid (erythroid burst-forming units; BFU-E) progenitors increased on day 3 to 220%-240% and 120%-130% of the control value, respectively. This was accompanied by a decline of the initial spleen colony-forming units (CFU-S) (day 8) pool size to approximately 60%. These patterns were similar in the bone marrow and the spleen. In addition, the tritiated thymidine kill of femoral and splenic CFU-S rose significantly (p less than 0.05) from 16% +/- 3% to 38% +/- 2% and from 3% +/- 1% to 17% +/- 2%, respectively. A sudden decline of peripheral reticulocytes between days 2 and 3 from 2.8% +/- 0.3% to 0.6% +/- 0.2% was observed, whereas the hematocrit gradually decreased from day 1 to day 4 from 45.2% +/- 0.1% to 39.3% +/- 0.3%. The white blood cells were not affected. From these results we conclude that stem cells were stimulated as a consequence of the suppression of the intermediate cell stages. As analyzed in the accompanying paper, this confirms a prediction stated by a quantitative theoretical concept of in vivo stem cell regulation.

In a recent theoretical model of stem cell regulation, specific quantitative assumptions were made about an in vivo feedback process from erythroid and granuloid precursor cell stages to the spleen colony-forming units (CFU-S), erythroid burst-forming units (BFU-E), and granulocyte-macrophage colony-forming units (CFU-GM). Utilizing specific effects of the antibiotic thiamphenicol (TAP), new experiments have been performed to challenge this model. Here these data are treated in an analysis that implies three steps. First, model assumptions about TAP toxicity are justified. The toxic TAP effects on erythroid and granuloid precursors are quantified as a continuous reduction of the normal amplification coefficient for CFU-E (down to 1/250), proerythroblasts, basophilic erythroblasts, and proliferating granuloid precursors (down to 1/4). Second, the original model predictions for the behavior of CFU-S, CFU-GM, and BFU-E are compared with the corresponding data. Third, discrepancies are discussed and it is demonstrated that adjustment of one single parameter resolves most of them. Thus one can quantitatively explain the experimental results for CFU-S, BFU-E, and CFU-GM by an activation of the regulatory process postulated: the decline in erythroid (and granuloid) cell numbers enhances the cycling of CFU-S while their self-renewal probability is reduced; consequently CFU-S numbers decline; as more cells differentiate towards BFU-E and CFU-GM per unit time the cell numbers of these cell stages increase. Thus the new data on stem cell behavior during TAP treatment support the hypothesis of a feedback from erythroid and granuloid precursors to the stem cells.

A mathematical model of erythropoietic cell production and its regulation process has been proposed in a preceding paper. It is primarily based on the assumption that the number of cell divisions taking place in the CFU-E and erythropoietic precursor stages is regulated depending on the oxygen supply of the tissue. Quantitative dose-response relationships for in vivo erythropoiesis are suggested. Here, we demonstrate that this model adequately reproduces data obtained in situations of stimulated erythropoiesis in mice and rats. In detail, this implies a quantitative description of the following processes: (1) Changes in tissue oxygen tension (Pto2) following removal of red cells (bleeding, haemolytic anaemia) or increase in plasma volume (dilution anaemia) or decrease in atmospheric oxygen pressure (hypoxia). (2) Pto2 dependent erythropoietin (EPO) production. (3) Dose-response of EPO on erythropoietic amplification (up to two to four additional mitoses). (4) The changes of the marrow transit time. Model simulations are compared with experimental data for changes of erythropoiesis during hypoxia, EPO-injection, and different forms of anaemia. A satisfactory agreement suggests that the model adequately describes and correlates different direct and indirect ways to stimulate erythropoiesis. It quantifies the role and relative contribution of the haematocrit, haemoglobin concentration, atmospheric oxygen pressure, tissue oxygen pressure, and plasma volume as triggers in erythropoietic stimulation under various conditions. Furthermore, the model may allow to optimize the scheme of EPO-administration and to find the maximum increase of erythropoiesis for a given amount of erythropoietin.

Although the amount of experimental data on the behavior of the hemopoietic system after various perturbations is considerable, a conclusive understanding of hemopoietic regulation is still absent. In the last years, we have examined murine erythropoiesis, thrombopoiesis, granulopoiesis, and stem cell hemopoiesis by means of mathematical modeling in order to identify some of the underlying principles. Our results can be summarized in four hypotheses. 1) The regulation of hemopoiesis is governed by three interrelated control loops: autoregulation of stem cells, feedback from progenitors and precursors to the stem cells, and feedback from mature cells to progenitor and precursor cells. 2) The feedback from mature cells to the progenitor and precursor cells predominantly varies the number of cell divisions taking place during hemopoietic maturation. 3) Two distinct properties of the stem cells are regulated: their cyclic activity and their self-renewal. Both are under the control of stem cell autoregulation and the feedback from progenitors and precursors. 4) A large variance in the maturation time from the stem cells to the mature cells stabilizes the hemopoietic control. The mathematical formulation of these assumptions allows us to understand a broad range of experimental observations including recovery from stem cell damage, hypoproliferative and hyperproliferative situations, and interactions between different cell lines.