Leukemia Research Vol. 14, No. 6, pp. 495-499, 1990. 0145-2126/90 $3.00 + .00 Printed in Great Britain. Pergamon Press plc

OPEN FORUM

MODELS OF HAEMOPOIESIS

GEOFFREY BROWN, CHRISTOPHER M. BUNCE and JANET M. LORD

Department of Immunology, University of Birmingham, U.K.

(Received 31 January 1990. Accepted 4 February 1990)

Abstract--To date, various models have been proposed to explain the diversification of haemopoietic stem cells along one of at least six pathways of differentiation. Consideration of evidence for and against particular models leads to the conclusion that a precise lineage map for the haemopoietic system is, as yet, unavailable. However, recently available cell and molecular biology techniques provide the means to resolve this problem.

Key words: Haemopoiesis, stem cells, lineage determination, differentiation, leukaemia, protein regulators.

A PRECISE lineage map for the haemopoietic system which is, as yet, unavailable, is important for a num- ber of reasons. The map would provide a basis essen- tial to developing an understanding of the regulation of cell numbers within stem cell and committed pro- genitor cell compartments. The inter-relationship between haemopoietic lineages also affects con- sideration of cell involvement in malignant trans- formation. Furthermore, since leukaemia may result from perturbations in intracellular processes which integrate cell proliferation with maturation [1], the manner in which differentiation options are regulated within cells is pertinent to resolving imbalances in malignancy.

A lineage map which is most often used to describe the haemopoietic system is shown in Fig. 1. The haemopoietic stem cell gives rise to lymphoid pro- genitor cells and multipotent progenitor cells with the potential for granulocyte, erythrocyte, macrophage and megakaryocyte differentiation (GEMM-CFC). This particular divergence of potentials (see Fig. 1, pl/2) appears to be a first step in the generation of progenitor cells [2]. Subsequently, the GEMM-CFC give rise to progenitor cells with the potentials for either erythroid or megakaryocyte or granulocyte

Abbreviations: GEMM-CFC, granulocyte, erythrocyte, macrophage and megakaryocyte colony forming cell; GM- CFC, granulocyte, macrophage colony forming cell; IL-3, interleukin 3; GM-CSF, granulocyte, macrophage colony stimulating factor; M-CSF, macrophage colony stimulating factor; G-CSF, granulocyte colony stimulating factor.

Correspondence to: G. Brown, Department of Immu- nology, The Medical School, University of Birmingham, Birmingham B15 2TJ, U.K.

495

and macrophage differentiation. The restriction in these differentiation potentials is postulated to occur in a progressive and stochastic manner [3]. Thus, the lineage map does not reveal a single principle which governs the diversification of progenitor cells since both ordered and stochastic processes restrict dif- ferentiation options. In other words, the model sug- gests that at least two deterministic mechanisms operate within cells of the haemopoietic system and that choices governed by ordered or stochastic events occur in a particular order (see Fig. 1 pl /2 followed by p2a/p2b/p2c). This could be the case, however, there is no clear rationale as to why the generation of lymphoid progrenitor cells and the association of potentials for granulocyte and monocyte differ- entiation (see Fig. 1 p2a/p2b) occurs in an ordered manner in contrast to the stochastic manner in which progenitor cells are restricted to differentiation towards erythrocytes, megakaryocytes and granulo- cytes/macrophages.

An alternative model for haemopoiesis is that the haemopoietic stem cell is strictly multipotent, in that receptors for all of the growth and development factors are expressed simultaneously, such that the stem cell can differentiate directly along any one of six or more pathways of maturation [4, 5] (see Fig. 2). In this case, the development along a particular pathway is influenced by the availability of and inter- action with one or more factors [4]. As argued pre- viously by Holtzer, the proposal of a pluripotent haemopoietic stem cell raises questions as to prin- ciples of cell diversification and also at a molecular level [6]. Data from various embryological studies

o y

G. BROWN et al.

Pla // Plb

GM - CFC

496

T Cells

O Stem Cell

Pl / P2

P2a/P2t

G

GEMM- CFC

~P2c

P2aa/ P2ab

/ \

B Cells Monocytes Granulocytes Erythrocytes Megakaryocytes

FIG. 1. Conventional representation of decision points during the diversification of haemopoietic progenitor cells. Differentiation pathway options are designated pl, p2, etc. GEMM-CFC and GM-CFC are colony forming cells restricted to granulocyte/erythrocyte/macrophage/mega- karyocyte differentiation and granulocyte/macrophage

differentiation respectively.

... ........... 0 " " p4 / ~ G,,..~o~yt,,

+

© Erythtocyte$

FIG. 2. A multipotent haemopoietic stem cell in a strict sense. Differentiation options pl ~ 5 are simultaneously

and equally available.

argue that cells are only capable of binary decisions such that a given cell set, undergoing one cell cycle, can generate a maximum of two different sets of cells. Thus, in 1979, Holtzer concluded that "I do not at this time believe that any cell is pluripotent".

Recent work concerning the nature of intracellular

signalling pathways which effect the growth and dif- ferentiation of cells presents difficulties as to the molecular events required for lineage restriction of pluripotent cells. There are a relatively small number of signalling processes within cells and binding of a variety of ligands to receptors generates the same second messengers. For example, common signals are generated by various haemopoietic growth fac- tors (IL-3, GM-CSF, M-CSF and G-CSF) leading to activation of the sodium/hydrogen antiport and intracellular alkalinization [7]. If the haemopoietic stem cell is truly multipotent, all the genetic elements and protein regulators which govern lineage deter- mination should be available for activation or, at least, can be rendered accessible. With a limited number of signalling pathways it is difficult, at present, to envisage how a particular extracellular signal or combination of signals can be transduced appropriately so as to elicit differentiation along only one of the number of pathways of differentiation available. As to this problem, the number of dif- ferentiation potentials that are available is an im- portant consideration. Recently, Herzenberg and

Stem cell

Models of haemopoiesis

Megakaryocytes Erythrocytes Granulocytes Monocytes S cells T cells

P1

FIG. 3. An ordered process of progenitor cell development. Differentiation options P1 ~ 6 are expressed individually and in a preferred order. Cells gradually acquire one dif- ferentiation potential followed by loss of this capacity as

the next differentiation option is expressed.

497

Herzenberg have argued that there are at least two distinct developmental pathways for B cells [8]. Increases in the number of distinct lineages of B cells, T cells and myeloid cells would increase the difficulty of providing a mechanistic explanation of com- mitment if a multipotent haemopoietic stem cell has also various B lineage developmental options avail- able concomitantly.

A further model for haemopoiesis postulates that lineage potentials are expressed individually, sequen- tially and in a predetermined order during the devel- opment of progenitor cells, such that only one or two differentiation options are available at any given time [9, 10] (Fig. 3). As to the consideration of a mechanism for the sequential expression of lineage potentials, stem cells may undergo a series of quantal cell divisions during which differentiation options arise individually and sequentially. Alternatively, the process of availability and loss of differentiation options could occur without the stem cells leaving GO and entering cell cycle. In this case, differentiation options would be available transiently such that as cells lose one potential they acquire the next.

The evidence which supports a sequential model of lineage determination is the particular close phenotypic relationships between haemopoietic cells committed to maturation along various pathways of differentiation. Analyses of patterns obtained for total cellular phosphoproteins, by 2-dimensional gel electrophoresis, has revealed few differences in phosphoproteins constitutively phosphorylated within cell lines which typify precursors of erythro- cytes, granulocytes, monocytes and B lymphocytes. Densitometric analyses of changes in the phospho- rylation status of proteins which differ between the cell lines reveals a gradual decrease or increase in the phosphorylation status of proteins such that the lines

can be arranged in a linear sequence of relatedness. Thus, a line with the potential for erythroid dif- ferentiation was shown to be most closely related to lines able to differentiate towards neutrophils, which most closely relate to lines able to differentiate towards monocytes which, in turn, are related to pre-B cell lines [11, 12]. The interpretation of these gradual phenotypic changes is that progenitor cells with the above lineage potentials have diversified initially in a progressive and linear manner. A similar argument supported by other data is as follows. Stud- ies of the growth of normal bipotent cells in semi-solid medium has clearly established the close relationship between cells with the potentials for neutrophil and monocyte differentiation. Diverse observations sug- gest that monocyte and B lymphocyte progenitor cells are also closely related [13-19]. Placing the progenitor cells for neutrophils, monocytes and B cells along a linear development pathway, rather than along three distinct pathways of cell diversification, would explain the above two relationships.

A linear sequence of progenitor cell development readily explains various haemopoietic disease states. These include the spectrum of cell involvement seen in the myelodysplastic and myeloproliferative dis- orders [20,21], immunological abnormalities in myelodysplasia [22, 23], the progression of chronic myeloid leukaemia to a B lymphoid blast crisis [24] and the co-existence of myeloproliferative disorders and lymphomas [21] and of myelodysplastic syn- drome and acute lymphoblastic leukaemia [25]. Finally, a preferred course of expression of lineage potentials would infer a developmental program within cells. Arguments in favour of a linear program of cell diversification, derived from studies of a var- iety of organisms, have been considered elsewhere [26].

498 G. BROWN et al.

At present, the arguments for and against par- ticular models for haemopoiesis are based on circumstantial evidence. The difficulty as regards pro- viding direct evidence is one of enumerat ing the number of lineage options within a stem cell at a given time point, in other words, without the cell dividing to give rise to diverse progeny. The avail- ability of a large number of antigenic markers for haemopoiet ic cells at various stages of maturat ion allows the possibility to define and purify haemo- poietic stem cells. Molecular markers of differ- entiation options, as to gene regulators whose expression is prerequisite for cells to differentiate along a particular pathway, should be revealed by now extensive studies of proteins which bind to DNA. Enumera t ion of the number of protein regu- lators of distinct lineage options which are simul- taneously expressed within a single haemopoietic stem cell is most likely to require amplification of cell constituents to a level appropriate to phenotypic studies. For example, the polymerase chain reaction could be used to amplify m R N A from a single stem cell for analyses of the presence or absence of par- ticular m R N A species to assess simultaneous tran- scription of various protein regulators. Hence, whether the haemopoiet ic stem cell is multipotent in a strict sense or has only one or two differentiation options available at any given time point could be resolved, in the near future, by the use of currently available cell and molecular biology techniques.

Acknowledgements--Research in our laboratory is sup- ported by the Leukaemia Research Fund and The Royal Society. J.M.L. is a Royal Society 1983 University Research Fellow. We are grateful to Ruth Jarrett for helpful discussions during the preparation of this manu- script.

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