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The study of the process of haemopoiesis is rendered difficult by the fact that it is extremely difficult to distinguish or separate individual stem cells from their products during the earlier stages of differentiation. However, a picture of the process of differentiation is now beginning to emerge (Figure 6.1). During the haemopoietic process, the stem cells differentiate, producing cells that become progressively more restricted in their choice of developmental options.
The production of many mature blood cells begins when a fraction of the stem cells differentiate, forming a specific cell type termed CFU-S (CFU refers to colony forming unit). These, in turn, differentiate yielding CFU-GEMM cells, a mixed CFU which has the potential to differentiate into a range of mature blood cell types, including granulocytes, monocytes, erythrocytes, platelets, eosinophils and basophils. Note that lymphocytes are not derived from the CFU-GEMM pathway, but differentiate via an alternative pathway from stem cells (Figure 6.1).
The details of haemopoiesis presented thus far prompt two very important questions. How is the correct balance between stem cell self-renewal and differentiation maintained? And what forces exist that regulate the process of differentiation? The answer to both questions, in particular the latter, is beginning to emerge in the form of a group of cytokines termed ‘haemopoietic growth factors’ (Table 6.2). This group includes:
• several (of the previously described) interleukins (ILs) that primarily affect production and differentiation of lymphocytes;
• colony stimulating factors (CSFs), which play a major role in the differentiation of stem-derived cells into neutrophils, macrophages, megakaryocytes (from which platelets are derived), eosinophils and basophils;
Biopharmaceuticals: Biochemistry and Biotechnology, Second Edition. Gary Walsh John Wiley & Sons Ltd: ISBN 0 470 84326 8 (ppc), ISBN 0 470 84327 6 (pbk)
Table 6.1. The range of blood cells that are ultimately produced upon the differentiation of pluripotential stem cells (see text for details). (Note that osteoclasts are multinucleated cells often associated with small depressions on the surface of bone; they function to reabsorb calcified bone)
T and B lymphocytes Erythrocytes Monocytes Osteoclasts
Figure 6.1. A simplified overview of the haemopoietic process as currently understood. Refer to text for details. CFU = colony forming unit; BFU = burst forming unit
erythropoietin, which is essential in the production of red blood cells; thrombopoietin, which is essential in the production of platelets.
Most of these haemopoietic growth factors are glycoproteins, displaying a molecular mass in the region of 14-24 kDa. Most are produced by more than one cell type and several display redundancy in their actions. In general several such regulators can stimulate proliferation of any
HAEMOPOIETIC GROWTH FACTORS 257 Table 6.2. Major haemopoietic growth factors described to date
Granulocyte-macrophage-colony stimulating factor (GM-CSF) Granulocyte-colony stimulating factor (G-CSF) Macrophage-colony stimulating factor (M-CSF)
Leukaemia inhibitory factor (ILF)
one haemopoietic cell lineage. This is due to the presence of receptors for several such factors on their surface. Receptor numbers for any one growth factor are low (less than 500/cell) and proliferation can be stimulated even when only a small proportion of these are occupied.
Genetic engineering has allowed the production of recombinant forms of virtually all of the currently recognized haemopoietic growth factors. This has facilitated development of a greater depth of understanding of the haemopoietic process. When stem cells were treated in vitro with various growth factors, only IL-3 was able to sustain/promote their growth and differentiation. Treatment with individual CSFs, or other ILs, failed not only to promote differentiation but, in most cases, even to promote continued survival of the cells. Subsequent experimentation illustrated the requirement for a combination of growth factors in most instances. A combination of granulocyte-colony stimulating factor (G-CSF) and macrophage-colony stimulating factor (M-CSF) was found to promote neutrophil and macrophage differentiation, while similar synergistic interactions were noted when IL-1 and IL-3 or G-CSF and granulocyte-macrophage-colony stimulating factor (GM-CSF) were used in combination. Such requirements for stem cell maintenance and differentiation are also likely to be mirrored in vivo.
In vivo, haemopoietic cells are usually found clustered in close association with various types of bone marrow stromal cells. It appears that the stromal cells play a direct role in promoting proliferation/differentiation of the stem cells. Indeed, co-culture of the stromal and stem cells facilitates self-renewal and differentiation of the latter in the absence of exogenously added growth factors. Interestingly, direct contact between the two cell types is required. This appears to indicate that the growth factors are physically associated with the surface of the stromal cell, rather than being released in soluble form. When grown in vitro, stromal cells are known to produce various haemopoietic growth factors, including IL-4, -6 and -7, as well as G-CSF.