STEM CELL RESEARCH

Haematopoietic Stem Cells, Cytokines and Glycosylation

All mature blood and immune cells develop from a population of adult stem cells called haematopoietic stem cells (HSCs). HSCs are characterised by expression of the CD34 cell surface antigen. These CD34 positive (CD34+) cells have the capacity for self-renewal, are able to differentiate into a variety of specialised cell types and can mobilise out of the bone marrow and into the circulation. The regenerative capacity of these cells is finely controlled by the cellular environment, and through molecular signals from cytokines. HSCs can be isolated from bone marrow, umbilical cord blood or peripheral blood.

HSCs are currently the only type of stem cells approved for human use. Currently HSCs are used stem cell transplantation following myeloablative chemotherapy to treat haematological malignancies including leukaemia and lymphoma, as well as the treatment of inherited blood disorders such as anaemia and thalassaemia. Research is now focusing on using HSCs to treat autoimmune disorders such as diabetes and rheumatoid arthritis. When allogeneic HSCs are transplanted they appear to have anti-tumour activity and current research is focusing on HSC transplantation as part of non-myeloablative therapy regimes for malignancies^1,2

Generation of sufficient cell numbers of the appropriate cell type for therapeutic use requires stem cells to be expanded ex-vivo in cell culture. Ex-vivo expansion requires chemically defined, serum-free medium containing an optimal combination of cytokines and growth factors. These can include G-CSF, GM-CSF, M-CSF, EPO, TPO, IL-3, IL-6, SCF and Flt-3 ligand as well as other cytokines. Some of these are discussed below.

Many of the growth factors and cytokines used to culture and expand stem cells are highly glycosylated. Glycosylation is a species-specific post-translational modification that affects the biological properties of many cytokines. Recombinant proteins produced from E. coli are not glycosylated, whereas cytokines produced in different expression systems such as insect, yeast or other mammalian cells exhibit different glycosylation patterns compared to native human proteins. The importance of human-specific glycosylation of cytokines is now being recognised, as differences in glycosylation can result in different biological properties of a protein.

EPO

Erythropoietin (EPO) is one of the most-studied molecules in terms of the necessity of glycosylation for protein function. EPO induces erythroid differentiation of CD34+ cells, leading to the generation of red blood cells in vivo. EPO is currently used to treat anaemia resulting from kidney disease or chemotherapy. EPO is heavily glycosylated with one O-linked and 3 N-linked glycan sites, and an average oligosaccharide content of 40% by weight. Glycosylation of EPO is important for secretion from the cell, conformation, solubility, stability and biological activity³. Until recently most commercially available recombinant human EPO was purified from Chinese Hamster Ovary (CHO) or Baby Hamster Kidney (BHK) cells. However, some human-specific oligosaccharides are not synthesised by CHO or BHK cells as they lack several sugar-transferring enzymes found in human cells⁴. CHO and BHK expressed EPO also have Galβ 1-4GlcNac (LacNAc) repeats not found on native human EPO. These differences could result in differences in the biological activity of EPO produced in non-human systems such as CHO cells⁵.

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G-CSF

Granulocyte colony stimulating factor (G-CSF) regulates the differentiation and maturation of cells of the granulocytic lineage. G-CSF has one O-linked glycan and glycosylation has been shown to increase the stability of G-CSF⁶, resulting in higher biological activity and greater ability to stimulate CD34+ stem cell proliferation⁷. G-CSF has been used extensively to mobilise stem cells from cancer patients and normal donors, producing peripheral blood progenitor cells which, when transplanted, mature into monocytes, neutrophils platelets and erythroid cells⁸. Mobilisation with glycosylated G-CSF was associated with normal neutrophil morphology and function^9,10. Conversely stem cell mobilisation with non-glycosylated rhG-CSF resulted in neutrophils with defective chemotaxis and increased actin polymerisation, emphasising the importance of glycosylation on therapeutic proteins.

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SCF

Stem Cell Factor (SCF) is a glycoprotein that augments the proliferation of erythroid, myeloid and lymphoid haematopoietic progenitors in bone marrow culture. SCF acts synergistically with other cytokines. It is known to protect progenitor cells from apoptosis¹¹ and also primes stem cells for differentiation. SCF has been used clinically in combination with G-CSF to mobilise CD34+ cells into the circulation of cancer patients. SCF is heavily glycosylated, with N- and O-linked glycans on multiple residues. Unlike EPO and G-CSF, the effect of glycosylation on the biological activity of SCF has not been clearly elucidated.

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TPO

Platelets are produced by fragmentation of precursor cells called megakaryocytes, which mature from haematopoietic stem cells in the bone marrow. Thrombopoietin (TPO) is the major growth factor regulating megakaryopoiesis and platelet production. TPO consists of 2 domains, the N-terminal domain is highly homologous to EPO and is involved in receptor binding, while the C-terminal domain is highly glycosylated and required for both secretion¹² and stability of TPO¹³. TPO has been used successfully to treat thrombocytopaenia following low-dose chemotherapy, but has been less successful in regimes following high-dose chemotherapy and in stem cell transplants¹⁴. More research is required before TPO can be used successfully in ex-vivo stem cell expansion regimes.

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GM-CSF

Granulocyte-macrophage colony stimulating factor (GM-CSF) stimulates HSCs to become granulocytes or monocytes. Native human GM-CSF purified from activated lymphocyte conditioned medium shows a broad range in the level of glycosylation, with high, low and intermediate glycoforms¹⁵. Glycosylated GM-CSF has a longer plasma half-life than non-glycosylated forms of GM-CSF¹⁶. GM-CSF is used to expand stem cells ex-vivo, and also to mobilise CD34+ cells in stem cell donors. It is currently being trialled as an adjunct to chemotherapy and immunotherapy, to prevent graft-versus-host reactions, and to enhance the ADCC response to therapeutic antibodies in mouse models by increasing the activity mediated by donor NK cells prior to infusion.

In view of the evidence showing how strongly glycosylation affects a protein’s function in vitro and in vivo, it is therefore of paramount importance to select a source of recombinant cytokines that have the correct glycosylation for all research and clinical applications.

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References

1. Joshi et al. (2000) Clin. Can. Res. 6 (4351-4358)
2. Resnick et al. (2005) Transplant Immunology. 14 (207-219)
3. Egrie and Brown (2001) Nephrol. Dial. Transplant. 16 suppl. 3 (3-13)
4. Skibeli et al. (2001) Blood. 98 (3626-3634)
5. Cointe et al. (2000) Glycobiology. 10 (511-519)
6. Carter et al. (2004) Biologicals. 32 (37-47)
7. Qerol et al. (1999) Hematologica. 84 (493-498)
8. Anderlini and Champlin (2002) Drugs. 62 suppl. 1 (79-88)
9. Azzara et al. (2001) Am. J. Hematol. 66 (306-307)
10. Carulli et al. (2006) Am J Hematol. 81 (318-323)
11. Zeuner et al. (2003) Blood. 102 (87-93)
12. Muto et al. (2000) J. Biol. Chem. 275 (12090-12094)
13. Kuter and Begley (2002) Blood. 100 (3457-3469)
14. Tijssen et al. (2006) Transfusion Med. Reviews. 20 (283-293)
15. Cebon et al. (1990) J. Biol. Chem. 265 (4483-4491)
16. Okamoto et al. (1991) J. Biol. Chem. 286 (562-568)

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