Growth Factors and Cytokines

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Introduction Growth Factors and Cytokines: Classification and Nomenclature Epidermal Growth Factors (EFG) and NRG Family Fibroblast Growth Factor Family (FGF) Hematopoietic Cytokines	Growth Factors in Stem Cell Biology Hepatocyte Growth Factor (HGF) Insulin-like Growth Factors (IGF) Interleukin family (IL) Neurotrophic Factors Platelet Derived Growth Factors (PDGF) Transforming Growth Factor (TGF) Superfamily	Tumor Necrosis Factor (TNG) Superfamily Applications of Cytokines and Growth Factors in Cancer Research Vascular Endothelial Growth Factors (VEGF) Fetal Bovine Serum

Introduction

Jennifer Fries

Technical Marketing Specialist, Cell Culture

The importance of cytokines and growth factors in cell culture has been demonstrated time and time again since the inception of cell culture. Beginning with the first successful efforts of growing cells in culture, there have been so called 'secret ingredients' added to culture medium that have, over time, given rise to a the belief that cell culture is as much of an art as a technique. Serum and other < extracts and the painstaking, continued attempts to characterize them have greatly contributed to this situation.

Studies published as early as 1921 acknowledged the fact that there were critical unknowns that were essential for normal growth, metabolism, and development of cells in culture. Zilva, Goldblatt, Sanford and a number of other cell biology pioneers were the first to admit that there were unknown factors influencing the general health of both cellular and animal models.

Over time, we have learned that many of these unknown factors are nutrients such as vitamins, amino acids, sugars, albumins and transferrins. However, even when all the necessary nutrients are present certain cells especially primary cells do not proliferate. Serum seemed to provide the unknown factors that encouraged cell proliferation. Today we know the agents that are responsible for cell proliferation and differentiation are growth factors and cytokines.

Growth factors can be described as proteins that bind to receptors on the cell surface of non-hematopoietic cells and result in proliferation or differentiation of the affected cells. Each family of growth factors affect specific cell types. For example, epidermal growth factors (EGF), affect epithelial cell types, similarly platelet derived growth factors (PDGF), affect only fibroblasts commonly found in connective tissues.

Cytokines, often compared with growth factors, are a class of signaling molecules (proteins, peptides and glycoproteins) that affect primarily the cells of the immune system but can affect other diverse cell types outside of the immune system as well. Cytokines are generally thought of as part of the signaling mechanism that orchestrates the immune response to bacterial infection. The effects of cytokines on cells are varied, some like growth factors cause cell proliferation, others may cause chemotaxis between different cell types, and others can even cause apoptosis.

Cytokines and growth factors are somewhat similar in their structure and mechanism of action. Both bind to specific cell surface receptors that initiate signaling pathways and well as having receptors that share distinct structural homologies. Many growth factors and cytokines also share several intracellular signaling components through which the activated cell surface receptor transmits its message to the cell nucleus.

In both the research and pharmaceutical community, there is a growing need for defined serum-free media that eliminates the variability and the potential virus and prion contamination as well as facilitates the purification of recombinant proteins. The development of serum-free media will often necessitate the use of certain growth factors, and cytokines. This is always true for primary cell lines but often necessary for transformed cell lines and hybridomas as well.

Selecting the appropriate growth factors and cytokines for your application is an important task and can be often based on existing protocols or by chance. Growth factors and cytokines are critical to successful cell differentiation and proliferation. Within this issue of Biofiles, you will find Sigma's listing of cell culture tested cytokines and growth factors along with helpful information that will make chosing the products you need easier.

Sigma-Aldrich carries and extensive line of high quality, cell culture tested growth factors and cytokines to complement your research needs. Please visit us at sigma-aldrich.com/growthfactors to view our complete list on-line.

Classification and Nomenclature

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Growth factors and cytokines have historically been classified into 'families' based on their apparent activity and/or impact on a given cell type, system, or tissue. Lately however, there has been an effort to establish naming based upon growth factor and cytokine receptors. Growth factor and cytokine receptors are highly conserved, and by utilizing them as a key for developing a systematic naming process, the field of growth factor families has narrowed.

Naming based on the differentiation of the cytosolic receptor domains has resulted in several major 'families' of growth factors and cytokines. In addition, the extracellular domains of these receptors demonstrate considerable homologies. This degree of homology in the extracellular domain leads to the highly conserved structures of growth factors and cytokines. The conserved nature of these receptors accounts for multiple signal transduction pathways effecting or impacting similar processes.

The identification, origin, activity, and signal transduction pathways of growth factors and cytokines remains in the discovery phase, however some of the more established, influential, and common compounds have been characterized. In recent years, cell biologists have attempted to reach a consensus on naming and nomenclatures, however naming schemes previously have been rather chaotic, leaving some compounds in rather odd groups. For example, bone morphogenic proteins (BMP), which affect bone and cartilage formation, fall into the tumor growth factor-beta (TGF-b) super-family. Another example is the tumor necrosis factor (TNF) protein. It is not useful as an anti-tumor therapy, but is highly active in immune modulation and inflammation.

Cytokines are often referred to as “growth factors”, but the reverse is not necessarily the case. Historically, growth factors have been thought of as compounds that have a positive effect on cell growth and expansion while cytokines are typically considered to have an immunological or hematopoietic response. Cytokines such as interleukin-2 (IL-2), which promote long term growth of activated T cells and related cell types fit the 'growth factor' description, but are classified as cytokines for their immunological responses and molecules such as the FAS ligands, which are involved in the initiation of programmed cell death - certainly not a positive effect on cell growth and expansion -fall into the cytokine category.

Over the years these compounds have been categorized into various classes, families, and super families, including the bone morphogenic proteins, epidermal growth factors, fibroblast growth factors, interferons, transforming growth factors, tumor necrosis factors, and vascular endothelial growth factors. As new molecules and pathways are identified, the terms 'growth factor' and 'cytokine' have come to be used interchangeably.

View FGF Family Tables

Hematopoietic Cytokines

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Numerous cytokines are involved in the regulation of hematopoiesis within a complex network of positive and negative regulators. Some cytokines have very narrow lineage specificities of their actions, while many others have rather broad and overlapping specificity ranges. Listed within this section include the cytokines whose predominant action appears to be the stimulation or regulation of hematopoietic cells. The term ''colony stimulating factor'' (CSF) was a designation originally given to agents discovered to stimulate growth of colonies containing differentiated myeloid cells from single bone marrow-derived precursor cells plated in semisolid medium. The glycoproteins considered to be CSFs include granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), interleukin-3 (multi-CSF or IL-3), interleukin-5 (IL-5), erythropoietin (EPO), ¹ and thrombopoietin (TPO). ²

A number of other cytokines exert profound effects on the formation and maturation of hematopoietic cells, with most of these belonging to the structural class known as the ''4-α-helical bundle'' family of cytokines, ³ which include stem cell factor (SCF), flt-3/flk-2 Ligand (FL) and leukemia inhibitory factor (LIF). Other cytokines or ligands, such as jagged-1, transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) also play significant roles in modulating hematopoiesis. Descriptions and listings of IL-3, IL-5, TGF-β and TNF-α products are listed in other sections by name.

References
1. Brugger, W., et al., Clinical role of colony stimulating factors. Acta Haematol. , 86, 138-147 (1991).
2. Lok, S., and Foster, D., The structure, biology and potential therapeutic applications of recombinant thrombopoietin. Stem Cells , 12, 586-598 (1994).
3. Nicola, N., An introduction to the cytokines, in Guidebook to Cytokines and Their Receptors, Nicola, N., ed., Oxford Press (New York, N.Y.: 1994), pp. 1-7.

Cells derived from the culture of neural stem cells grown in the presence of EGF ( Cat. No. E9644 ) and LIF ( Cat. No. L5283 ). The cells were expanded in ( Cat. No. S3194 ) and then moved to conditions to allow them to differentiate. This image shows differentiated cells fixed and stained with an antibody for GFAP (an astrocyte marker in green, Cat. No. G9269 ). The cells are also stained with ( Cat. No. P1951 and Cat. No. D8417 ).

View Hematopoietic Cytokine Tables

Growth Factors in Stem Cell Biology

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Growth factors are naturally occurring regulatory molecules, which bind to receptors on the cell surface. They stimulate cell and tissue function through influencing cell differentiation by changing their biochemical activity and cellular growth, and regulating their rate of proliferation. Numerous families of growth factors have already been identified and remarkable advancements have been made in understanding the pathways growth factors use to activate cellular proliferation and differentiation.

Medical researchers recognize the important role growth factors play in stem cell therapy and regenerative medicine. Stem cell research has the potential to dramatically change the treatment of human disease and serious injuries by providing a platform for deciphering the secrets surrounding the cellular processes controlling development, aging, and tissue regeneration. Regenerative medicine shows promise for repair of damaged tissues and organs, deliver safer and more efficient drugs, better disease models, and cures for numerous devastating diseases.

Stem cell treatment could put an end to inefficient disease treatments and lack of organ donations for organ transplants. Much is expected from Embryonic Stem Cell (ESC) research, due to their ability to differentiate into all possible cell types in the body and their potentially unlimited capacity for self-renewal. However, no approved medical therapies have been developed using ESC, due to the lack of understanding these cells and how are they influenced. Once growth factors are added to pluripotent stem cells under certain conditions, they are able to direct differentiation into three different germ layers, endoderm, mesoderm, and ectoderm, from which various cell types are derived. Endodermal cells develop into the liver and pancreas, mesodermal cells give rise to muscles and red blood cells, and ectodermal cells become the brain and skin. Tremendous progress has been made in understanding which growth factors manipulate the different lineage pathways. However, complete control of stem cell differentiation, from pluripotent to fully specialized cell, needs further investigation since it requires multiple growth factors in defined order and quantity and at defined time intervals.

A recent advancement in the field of regenerative medicine is the production of induced pluripotent stem (iPS) cells. iPS cells are embryonic-like stem cells, derived from reprogrammed adult cells. The use of iPS cells in place of embryonic stem cells from human embryos provides an avenue for creating an unlimited supply of embryonic-like stem cells generating a tremendous enthusiasm in this field. Induced pluripotent stem cell technology has enormous possibilities for safe treatment of numerous diseases, bypassing the current ethical and political issues of embryonic stem cells. Also, iPS cells allow for personalized medicine as both the nuclear and the mitochondrial DNA matches the donor. Furthermore, using iPS cells would not require any immune suppression, making them a superior choice over donor embryonic stem cells for therapeutic uses. Researchers are daily enhancing their already significant understanding of how growth factors affect both somatic and embryonic stem cell expansion and differentiation. However, they have only begun to explore the effects of growth factors on iPS cells and how they differ, if at all, from embryonic stem cells. Once they can completely control the fate of pluripotent stem cells, which is influenced by a number of cellular signals including growth factors, they will be able to direct these cells, both in vivo and in vitro, to become the specialized cells that make up all the tissue in the body and enable subsequent use in cell-based therapies, drug development, and disease modeling.

View Enlarged Image

Cells derived from the culture of neural stem cells grown in the presence of EGF (Cat. No. E9644) and LIF (Cat. No. L5283). The cells were expanded in (Cat. No. S3194) and then moved to conditions to allow them to differentiate. This image shows differentiated cells fixed and stained with an antibody for GFAP (an astrocyte marker in green, Cat. No. G9269). The cells are also stained with (Cat. No. P1951 and Cat. No. D8417).

View TGF Superfamily Tables

Product Description	Source	Recombinant Host	Physical Form	Assay	Cell Type Affected	Product Number
Tumor Necrosis Factor-α	human	yeast	buffered aqueous solution	-	osteoclast human fibroblast PMN cell	T0157
Tumor Necrosis Factor-α	from rat	Escherichia coli	powder	The biological activity is measured in a cytotoxic assay using a TNF-susceptible mouse L929 cell line in the presence of actinomycin D.	PMN cell human fibroblast osteoclast	T5944
Tumor Necrosis Factor-α	from mouse	Escherichia coli	powder	-	human fibroblast osteoclast PMN cell	T7539
Tumor Necrosis Factor-α	human	Escherichia coli	powder	The biological activity of TNF-α is measured by the cytolysis of murine L929 cells.	PMN cell osteoclast human fibroblast	T6674

Product Description	Source	Recombinant Host	Physical Form	Assay	Cell Type Affected	Product Number
Tumor Necrosis Factor-β	human	Escherichia coli	powder	The biological activity is measured in a cytotoxicity assay usin the TNF-suspectible murine L929 cell line in the presence of actinomycin D.	PMN cell human fibroblast osteoclast	T7799

	F2442	F6178	F4135	F0392	12107C	F0643	F3018	F6765
Source	bovine	bovine	bovine	bovine	bovine	bovine	bovine	bovine
Country	USA	USA	USA	USA	USA	USA	USA	USA
Sterility	‡	‡	‡	‡	‡	‡	‡	‡
Performance	‡	‡	‡	‡	‡	‡	‡	‡
Cloning Assay	‡	‡	‡	‡	‡	‡	‡	‡
Virus (raw material)	‡	‡	‡	‡	‡	‡	‡	‡
Mycoplasma	‡	‡	‡	‡	‡	‡	‡	‡
Bacteriophage	‡	‡	‡	‡	‡	‡	‡	‡
Endotoxin (EU/mL)	≤10	≤25	≤10	≤10	≤10	≤10	≤10	≤10
Hemoglobin (mg%)	≤20	≤25	≤20	≤20	≤20	≤20	≤20	≤20
Total Protein (g%)	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5	3.0 – 4.3	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5
Electrophorectic Pattern	‡	‡	‡	‡	‡	‡	‡	‡
IgG	‡	‡	‡	‡	‡	‡	‡	‡
Hormone Testing	‡	‡	‡	‡	‡	‡	‡	‡
pH at RT	6.7 – 8.0	6.7 – 8.0	6.7 – 8.0	6.7 – 8.0	6.8 – 8.1	6.7 – 8.0	6.7 – 8.0	6.7 – 8.0
Osmolality (mOsm/Kg H²O)	260 – 340	260 – 340	260 – 340	260 – 340	280 – 330	260 – 340	260 – 340	260 – 340
Chemical Analysis	‡	‡	‡	‡	‡	‡	‡	‡
Tetracyline	‡	‡	•	•	•	•	•	‡
‡ – Indicates testing is performed and product meets specification. • – Test is not performed. ^a – Available in Europe only. Please check with your local supplier regarding availability. ^b – Available in Canada only.

	F7524^a	F9665^a	F1051^b	F9423^c	12003C	12007C	12203C	12306C	F0926
Source	bovine	bovine	bovine	bovine	bovine	bovine	bovine	bovine	bovine
Country	Not of USA Origin	Not of USA Origin	Canada	Australia	Australia	Australia	New Zealand	USDA Approved	USDA Approved
Sterility	‡	‡	‡	‡	‡	‡	‡	‡	‡
Performance	‡	‡	‡	‡	‡	‡	‡	‡	‡
Cloning Assay	‡	‡	‡	‡	‡	‡	‡	‡	‡
Virus (raw material)	‡	‡	‡	‡	‡	‡	‡	‡	‡
Mycoplasma	‡	‡	‡	‡	‡	‡	‡	‡	‡
Bacteriophage	•	•	•	‡	‡	‡	‡	•	•
Endotoxin (EU/mL)	≤10	≤10	≤10	≤10	≤10	≤10	≤10	≤10	≤10
Hemoglobin (mg%)	≤20	≤20	≤20	≤20	≤25	≤25	≤25	≤25	≤25
Total Protein (g%)	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5	3.0 – 4.5
Electrophoretic Pattern	‡	‡	‡	‡	‡	‡	‡	‡	‡
IgG	‡	‡	‡	‡	‡	‡	‡	‡	‡
Hormone Testing	•	•	‡	‡	•	•	•	•	•
pH at RT	6.7 – 8.0	6.7 – 8.0	6.7 – 8.0	6.7 – 8.0	6.8 – 8.1	6.8 – 8.1	6.8 – 8.1	6.8 – 8.1	6.8 – 8.1
Osmolality (mOsm/Kg H²O)	260 – 340	260 – 340	260 – 340	260 – 340	260 – 330	260 – 330	260 – 330	260 – 330	260 – 330
Chemical Analysis	‡	‡	‡	‡	‡	‡	‡	‡	‡
Tetracycline	•	•	•	•	•	•	•	•	‡
‡ – Indicates testing is performed and product meets specification. • – Test is not performed. ^a – Available in Europe only. Please check with your local supplier regarding availability. ^b – Available in Canada only. ^c – Available in Japan only.

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Growth Factors and Cytokines