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Stem Cells, Vol. 16, No. 5, 314-321, September 1998
© 1998 AlphaMed Press

The Molecular Control of Hematopoiesis: Progress and Problems with Gene Manipulation

D. Metcalf

The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia

Key Words. Hematopoietic regulators • Transgenic • Gene inactivation

Correspondence: Dr. Donald Metcalf, The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, 3050 Victoria, Australia.


   Abstract
Top
 Abstract
Introduction
 Pseudotransgenic or Transgenic...
 Gene Inactivation Approaches
 Megakaryocytopoiesis
 References
 
The in vitro-based discovery and characterization of hematopoietic regulators were of great value in identifying many of the agents active in controlling hematopoiesis. Subsequent in vivo studies have validated most of the information obtained from the in vitro studies, although the in vitro studies proved to be somewhat misleading in predicting which agents would exhibit the greatest quantitative effects in vivo.

Establishing more clearly the actual situation in vivo has required a return to more complex, and often less satisfactory, studies on genetically manipulated whole animals. Of the two possible general approaches, gene inactivation models have proved more informative than transgenic, overexpression models. Each model has raised multiple questions in need of further resolution and the deletion studies have also indicated that other regulators must exist for various lineages, but have yet to be discovered. Of particular interest is the finding from gene inactivation studies that both G-CSF and thrombopoietin are necessary for the maintenance of normal numbers of progenitor cells in multiple lineages, suggesting that each of these lineage-dominant regulators may have broader actions when operating on cells in the stem cell and progenitor cell compartments.


   Introduction
Top
 Abstract
 Introduction
Pseudotransgenic or Transgenic...
 Gene Inactivation Approaches
 Megakaryocytopoiesis
 References
 
With the exception of early work on erythropoietin (Epo) and thrombopoietin (TPO), the discovery and initial characterization of the 20 or so regulators characterized as "hematopoietic" were based almost entirely on tissue culture studies, often not even using normal hematopoietic cells as target cells. The subsequent cloning of cDNAs for these regulators and the production of biologically active recombinant molecules allowed them to be tested in animals, and then some to be used successfully in clinical medicine.

Despite this satisfactory progress, the approaches used have resulted in a curious situation in which large areas of ignorance persist regarding the biology of these regulators. Are some regulators of more importance than others? Where exactly are they produced, by what cells and in response to what inductive stimuli? What is the usual fate of the regulator molecules? Why do some have significant actions on other tissues? Do these regulators control basal hemopoiesis or are they merely emergency control systems? How do regulators exert multiple actions on responding hemopoietic cells when only one type of receptor exists for each regulator? What determines whether a cell will respond by proliferation, maturation or functional activation? What disease states result from overproduction or deficiency of particular regulators? Why are there so many regulators with apparently similar actions on cells of a particular lineage? Is this evidence of redundancy or of complex, sophisticated, regulatory control? Are there aspects of hematopoiesis that cannot yet be accounted for by known regulators?

This is a formidable list of unresolved questions, most of which cannot be addressed using the most elegant reductionist cell cultures. It must also be admitted that many of these questions do not arouse the interest or approval of grant-giving committees or journal editors in a context where a particular agent has long since been in clinical use. Nevertheless, if hematopoiesis is to be understood, all of these questions need to be satisfactorily resolved.

In recent years, appreciation of the large gaps in present knowledge and advances in molecular biology have led to increasing use of gene manipulation to address some of these questions. After some initial comments on the utility of the various possible approaches, specific comments will be made on those regulators with actions on megakaryocyte and platelet formation.


   Pseudotransgenic or Transgenic Approaches
Top
 Abstract
 Introduction
 Pseudotransgenic or Transgenic...
Gene Inactivation Approaches
 Megakaryocytopoiesis
 References
 
Before the ready availability of regulators in recombinant form, it was felt that useful information on the biology of these regulators could be obtained by the study of mice exposed to continuous excess levels of these regulators. The biology of hematopoietic populations allowed a useful pseudotransgenic approach in which retroviruses containing the growth factor cDNA were used to infect marrow cells. A proportion of the cells integrated the growth factor cDNA under a viral long-terminal repeat promoter and the cells were used to repopulate irradiated recipients, resulting in an animal in which many of the hematopoietic cells were producing large amounts of the growth factor in a dysregulated manner.

This approach, involving cDNAs for GM-CSF [1], G-CSF [2], interleukin 3 (IL-3) [3], leukemia inhibitory factor (LIF) [4], Epo [5], IL-6 [6], flt3 ligand (FL) [7] and TPO [8], produced animals with extreme elevations of the respective factors and induced an extensive hyperplasia of those hematopoietic subsets known from in vitro studies to be responsive to the particular growth factor. With the reservation that the cellular source of the factor in these mice was not duplicating that in the normal body, the results certainly verified information on lineage-specific actions of the regulators and predicted accurately enough the likely adverse responses to the injection of excessive doses of these regulators.

Of particular interest for the present symposium, mice with hematopoietic cells over-producing TPO developed not only elevated platelet levels but also myelosclerosis [8], in line with earlier studies indicating a link between PDGF and hyperplasia of fibroblasts [9].

Possibly the most intriguing outcome of these experiments was the recognition that it could be very difficult to distinguish unrestricted cell proliferation from leukemia. Thus, in the presence of excess GM-CSF levels, proliferating populations of granulocyte-macrophage cells replaced much of the liver and lung with a fatal outcome, but such cells were not leukemic as assessed by their lack of autonomy in vitro or their failure to produce transplantable leukemias [1]. A similar difficulty was encountered in assessing the nature of the disease in mice with the polycythemia developing following sustained excess Epo levels [5]. Both situations were re-encountered in studies using bcr-abl [10] or activated Epo receptor [11], where it was recognized that true leukemic transformation could occur, but only after a long delay, and probably only after further mutations in the population.

Such extreme factor-induced hyperplasia is unlikely ever to be achieved using clinically administered growth factors and thus there is a minimal risk of leukemia induction with the usual use of such growth factors. Nevertheless, it is true that growth factors can expand and sustain populations that are already abnormal and at special risk of transformation, so that leukemia development can occur earlier and more frequently. This was demonstrated experimentally using immortalized FDC-P1 cells in GM-CSF transgenic mice [12] and may in part be the basis for acute myeloid leukemic transformation in some patients with congenital neutropenia with mutations in the G-CSF receptor gene [13].

Production of transgenic mice in which additional copies of growth factor genes are under the control of a strong independent promotor has provided animal models and results similar to those obtained using the pseudotransgenic approach. For example, GM-CSF [14], Epo [15] and G-CSF [16] transgenic mice show comparable hyperplasia of relevant hematopoietic populations.

It has become apparent that the transgenic approach can have serious flaws, based on the promoter chosen and the consequent site of expression of the transgene. This can result in quite odd, and probably misleading, tissue changes. For example, overexpression of LIF by bone marrow-seeding FDC-P1 cells led to osteosclerosis as well as systemic effects of LIF, such as loss of adipose tissue, but not to a change in thymus morphology [4]. In contrast, transgenic LIF mice with a thymic stromal-restricted pattern of LIF expression developed lymphoid follicles and germinal centers in the thymus and abnormal populations of CD4+ CD8+ lymphocytes in the lymph nodes [17]. Does either set of experiments reveal the genuine biological actions of LIF in the body? They certainly demonstrate possible actions of LIF but one wonders whether these effects would occur if the LIF was being produced by the cells usually producing this factor or even in response to the injection of LIF in large doses. Certainly, discrepant information has resulted from a number of transgenic models that casts some doubt on the value of this approach for resolving many questions regarding regulator biology, unless transgene expression can be restricted more effectively to those cells normally producing the regulator.


   Gene Inactivation Approaches
Top
 Abstract
 Introduction
 Pseudotransgenic or Transgenic...
 Gene Inactivation Approaches
Megakaryocytopoiesis
 References
 
The reservations arising from the discrepant results using the transgenic approach have led to increasing use of mice with inactivation of genes encoding growth factors or their receptors. This approach does not perturb the pattern of cells usually producing the factor but of course can provide no information on the important questions of the consequences of general or local overproduction of a particular factor. As a general comment, it can be said that the gene deletion approach has yet to document the absolute redundancy of any growth factor, although it has sometimes revealed that consequent deficiencies are in nonhematopoietic rather than hematopoietic tissues. As a general caution, it also needs to be emphasized that initial information on the phenotype of such deleted animals is usually incomplete and will underestimate the true consequences of the deletion, if for no other reason than that the animals under study are usually housed in hyper-protected, pathogen-free, conditions.

Prototype examples of the consequences of gene deletion are provided by studies relating to G-CSF [18] and GM-CSF [19, 20] or its receptor [21]. Inactivation of the gene encoding G-CSF results in a fertile, viable animal with a selective 70% depletion of circulating neutrophils and a reduced capacity to mount neutrophil responses to challenge infections [18]. The data indicate that G-CSF is a quantitatively important regulator of neutrophil production under basal conditions and in emergency situations. What was unanticipated in these mice was that the G-CSF-deficient state also resulted in a 50% fall in progenitor cells of all lineages. This result indicates that G-CSF is also an important regulator in the formation of a wide range of progenitor cells by stem cells and the multilineage nature of this action is in accord with in vitro studies indicating that G-CSF, when combined with stem cell factor (SCF), does enhance the formation of progenitor cells of multiple lineages in developing blast cell colonies [22]. It is this action that may underlie the multilineage elevation of stem and progenitor cells in the peripheral blood following the injection of G-CSF [23, 24].

The consequences of inactivation of the gene encoding GM-CSF or the gene encoding ß-common chain of its receptor are almost the complete opposite of the G-CSF data. Again, such mice are fertile and appear in good health as young adults. However, there is no depletion of mature cells in the peripheral blood, with the exception that ß-common chain deletion also prevents IL-5 action and therefore results in an eosinopenia [21]. There is also no depletion of marrow or spleen cellularity or progenitor cell levels, leading to the conclusion that, despite the powerful proliferative actions of GM-CSF on granulocytes, monocytes and eosinophils in vitro, GM-CSF appears to play no essential role in regulating the formation of these cells in vivo under conditions of basal hematopoiesis [19, 20]. GM-CSF is not however a redundant regulator. Both GM-CSF and ß common chain knockout mice develop severe lung disease characterized by alveolar proteinosis with parabronchial infiltrates of T and B lymphocytes and often patchy areas of pneumonia. The provisional conclusion is that this state is due to hypofunction of the alveolar macrophages consequent upon failure of necessary activation by GM-CSF. The disease can be corrected in GM-CSF-/- mice by locally produced GM-CSF where a GM- CSF transgene has been inserted using a lung epithelial promoter [25] and, in some humans with alveolar proteinosis, the condition can be improved by the administration of GM-CSF.

The opposite syndromes exhibited by G-CSF-/- and GM-CSF-/- mice document the validity of the in vitro data, indicating that hematopoietic regulators are not simply cell proliferation stimuli but also have other important actions on hematopoietic cells, including functional activation of mature end cells.


   Megakaryocytopoiesis
Top
 Abstract
 Introduction
 Pseudotransgenic or Transgenic...
 Gene Inactivation Approaches
 Megakaryocytopoiesis
References
 
The detection of hematopoietic regulators able to stimulate the proliferation of murine megakaryocytic precursors in vitro began with the recognition that mitogen-stimulated T lymphocyte-conditioned medium exhibits this biological activity [26]. The major factor responsible for this activity was identified as Multi-CSF (IL-3) [27, 28], an agent able to stimulate the formation both of mature colonies (containing a small number of large polyploid cells) and immature colonies (usually of larger size and containing a range of acetylcholinesterase-positive cells with varying levels of ploidy and in some colonies, cells of other lineages, most often erythroid). The development of recombinant GM-CSF allowed higher concentrations of this agent to be tested in vitro and, at these higher concentrations, GM-CSF was also an effective proliferative stimulus for megakaryocyte colony formation [29]. The subsequent development of IL-6 and SCF allowed both to be identified as having a weak and variable capacity to stimulate some mature megakaryocyte colony formation [30, and Metcalf D, unpublished data]. Two other agents, LIF and IL-11, were identified as being without direct megakaryocyte colony-stimulating activity but as being able to potentiate megakaryocyte colony formation stimulated by IL-3 [31, 32].

When recombinant TPO was developed, it proved to be able to stimulate the formation in vitro of mature megakaryocytic colonies but these were neither numerous nor of large size ( Fig. 1) [33]. The most characteristic feature of TPO-stimulated cultures is the absence of colonies of other lineages. When TPO is combined with SCF or IL-3, a marked enhancement is observable both of mature and immature megakaryocyte colony formation. Figure 1 illustrates typical data from mouse bone marrow cell cultures indicating that, as a single agent, IL-3 is superior to any other agent in stimulating megakaryocyte colony formation.



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  		Figure 1. Megakaryocyte colony formation in individual agar cultures of 50,000 C57BL mouse bone marrow cells. The final concentrations of the stimuli used were: recombinant murine TPO 50 ng/ml, recombinant murine SCF 100 ng/ml and recombinant murine IL-3 10 ng/ml. Cultures were incubated for seven days and cell counts were performed at x 400 on fixed cultures stained for acetylcholinesterase then Luxol Fast Blue and hematoxylin. Open boxes are colonies composed wholly of mature cells, closed boxes are colonies containing both mature and immature acetylcholinesterase-positive cells. Figures in circles are the total numbers of colonies in the cultures.

 
The SCF/TPO combination provides the simplest result to interpret and indicates not only that TPO in vitro is usually incapable alone of stimulating the proliferation of less mature megakaryocyte precursors but is also capable only of stimulating one-third of available mature precursors. Most megakaryocytic precursors appear to need stimulation either by IL-3 as a single agent or by a combination of TPO with either SCF or IL-3.

With these data in mind, the results of injecting recombinant factors into mice were surprising. IL-3 was essentially inactive in elevating platelet levels [34] although it did increase tissue megakaryocyte numbers [35], as did injected GM-CSF [36]. On the other hand, the gp130-signaling trio of regulators, LIF, IL-6 and IL-11, all were capable of elevating platelet levels but only to a moderate 1.5- to 2-fold above baseline values [37-39]. In sharp contrast, injected TPO was able to elevate platelet levels sixfold above basal levels [40].

These in vivo results have now been supported by a variety of gene inactivation studies. Deletion of the gene encoding IL-3 or GM-CSF causes no fall in platelet levels [41, 19] nor does deletion of the gene encoding IL-6, the IL-11 receptor or LIF [42-44]. Again, in sharp contrast, deletion of TPO or its receptor (Mpl) leads to a 95% fall in platelet levels [45, 46] and to a major depletion of tissue megakaryocytes and megakaryocytic progenitor cells [46].

The results clearly document that TPO is required to sustain 95% of basal levels of platelet production and these studies have been extended to show that injected TPO strongly enhances recovery of platelet numbers following the induction of thrombocytopenia [47].

Various studies have been performed to determine what regulates the residual thrombopoiesis occurring in the absence of TPO. In this laboratory, injection of LIF or IL-6 into mice lacking TPO receptors was able to elicit platelet elevations that, in terms of fold-rise from baseline values, were as high as those achievable in normal mice [48]. This indicates that such agents do not need to act via TPO as had been suggested [49], but to act in an independent manner and potentially these could be responsible, in part at least, for the residual thrombopoiesis.

The gene inactivation data appear to be quite unambiguous but the changes observed have led to unresolved problems of a similar nature to those arising with G-CSF responses and G-CSF inactivation. Why is IL-3 which is highly active in vitro almost without action in vivo and, conversely, why is TPO, a weak in vitro agent, so active in vivo? With G-CSF responses, it has been shown that these really depend on coaction of G-CSF with SCF [50], a combination that is strongly synergistic for granulocyte stimulation in vitro by amplifying the formation of granulocytic progenitor cells [22]. IL-6, however, shows a similar strong potentiation synergy with SCF in vitro [51], yet injected IL-6 cannot induce elevations in neutrophil levels at all comparable to those achievable by G-CSF; thus a problem remains. Data of the type illustrated in Figure 1 suggest strongly that the situation with TPO could be the same as that with G-CSF. There are only two agents that strongly synergize in vitro with TPO: IL-3 and SCF. IL-3 appears not to be produced in normal animals [52], which again leaves SCF as the likely key synergistic factor responsible for the major stimulation of platelet formation that is induced by the injection of TPO.

The G-CSF and TPO knockout models both share another phenomenon in need of explanation—a major reduction in progenitor cells of all lineages. In the case of G-CSF, as noted above, it is known that G-CSF synergizes with SCF to greatly amplify the production of a variety of lineage-restricted progenitor cells from the less mature multipotential blast colony-forming cells. Elevated numbers of progenitor cells have been observed following the injection of TPO [53]. However, the nature of this response remains unclear because current experiments in this laboratory have failed to document any amplification by TPO of the SCF-stimulated formation of progenitor cells by blast colony-forming cells.

Analysis of precursor cell numbers in mpl-/- mice has revealed two quite distinct changes. As shown in Figure 2, these mice have a 50% reduction in the number of megakaryocyte-committed progenitor cells, and a 90%-95% reduction of megakaryocytes and platelets. This is in line with what could have been predicted from the in vitro action of TPO, even if coaction with SCF might also need to be involved in this terminal megakaryocyte precursor proliferation and cytoplasmic maturation.



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  		Figure 2. Boxed areas indicate the total numbers of megakaryocyte progenitor cells per 50,000 marrow cells, the total number of megakaryocytes per 10 high power fields of the bone marrow and platelet numbers per µl in eight-week-old mice with or without Mpl receptors. Figures in circles are the percent values in mpl-/- mice compared to mpl+/+ mice. Data are drawn to scale only within each category of observations.

 
What was quite unanticipated was that, as shown in Figure 3, these mice also show a 90% reduction in colony-forming units-spleen (CFU-S), an approximately similar reduction in preprogenitor cells—blast colony-forming cells responsive either to SCF [54] or FL plus LIF [55]—and a 50% reduction in other lineage-committed progenitor cells. In the face of this generalized reduction in lineage-committed progenitor cells, it is remarkable that the mouse achieves a normal marrow cellularity and composition, possibly by having these progenitor cells undergo one additional cell division. The major reduction in CFU-S and preprogenitor cells suggests that TPO may play an important, and irreplaceable, role in the generation of these cells by repopulating stem cells. This is a somewhat bizarre action for a regulator that exhibits such a stringent lineage-restricted action on more mature hematopoietic precursor cells. No information is available whether repopulating stem cells actually express receptors for TPO and it may be wise to consider the possibility that these deficiencies in early hematopoietic cells may not be based on a direct action of TPO, or even reflect normal hematopoiesis. Is it possible that severe thrombocytopenia somehow triggers an attempted correction in hematopoiesis that results in a reduction in early hematopoietic cells or even triggers a regulator cascade with inhibitory or toxic consequences for these cells?



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  		Figure 3. Boxed areas indicate the total numbers of day 12 CFU-S, uncorrected for seeding efficiency, the total numbers of SCF and FL plus LIF-responsive preprogenitor cells (blast colony-forming cells) and lineage-specific progenitor cells (from IL-3-stimulated cultures) per 105 bone marrow cells from eight-week-old mpl+/+ and mpl-/-mice. Figures in circles are the percent values in mpl-/- mice compared to mpl+/+ mice. CFU-S data, W. Alexander, personal communication. All boxed areas are drawn to the same numerical scale.

 
In view of the reports that injected TPO does elevate lineage-committed progenitor cell numbers [53], a direct action of TPO is more likely but there is a need to establish more clearly the action of TPO on early hematopoietic cells.

In studies in this laboratory, it has been documented in G-CSF and GM-CSF knockout mice that deletion of a major hematopoietic regulator does not result in any compensatory overproduction of regulators with potentially comparable actions on cells of the lineage affected. Similarly, no overproduction has been observed in mpl knockout mice of IL-3, IL-6, IL-11 or LIF [48]. The failure to observe such compensatory responses strongly suggests an absence of any cell number-monitoring system, or at least one able to increase the production of potentially useful alternate regulators.

This conclusion is at odds with the view that platelet numbers do regulate TPO production. Certainly, there is an inverse relationship between platelet numbers and TPO levels [56] and receptor-mediated removal by platelets of TPO does influence the plasma half-life of TPO [57]. However, this does not prove the existence of a platelet-counting mechanism that activates regulator production, and it has been reported that most TPO production appears to be constitutive and invariant [58]. If such a sensor system exists, it appears not to be a major determinant of the production of potential replacing factors such as IL-6 or LIF.

Although the TPO and mpl knockout models have clearly established that TPO is the major regulator of platelet formation, it is also evident that our understanding of the biology of TPO remains incomplete. Further studies on these gene inactivation models should be able to resolve some of these unanswered questions.


   Acknowledgments
 
The work from the author's laboratory was supported by the Carden Fellowship Fund of the Anti-Cancer Council of Victoria, the National Health and Medical Research Council, Canberra and the National Institutes of Health, Bethesda, Grant No. CA-22556.

From THROMBOPOIETIN: FROM MOLECULE TO MEDICINE. STEM CELLS 1998;16(suppl 2):1-9. Contact AlphaMed Press for information about this supplement.


   References
Top
 Abstract
 Introduction
 Pseudotransgenic or Transgenic...
 Gene Inactivation Approaches
 Megakaryocytopoiesis
 References
 

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