Recombinant human CNTF protein (Qk063)

Ciliary Neurotrophic Factor (CNTF) is a member of the IL-6 family of cytokines. CNTF plays a crucial role in developing and maintaining the nervous system, in particular the optic nervous system. It promotes the maintenance, differentiation, and survival of various neurons, glial cells, and retinal cells.

CNTF has been used in vitro to initiate neural induction and differentiation. CNTF can be used to culture primary neurons and glial cells such as astrocytes and Schwann cells. It is also used to culture retinal cells and adipocytes.

Human CNTF has a molecular weight of 22.8 kDa. This protein is carrier-free, tag-free, and non-glycosylated to ensure its purity with exceptional lot-to-lot consistency. Qk063 is suitable for the culture of reproducible and high-quality neurons and other relevant cells.

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1000µg will be despatched as 2 x 500µg

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Summary

  • High purity species protein (Uniprot number: P26441)

  • >98%, by SDS-PAGE quantitative densitometry

  • Source: Expressed in E. coli

  • 22.8 kDa monomer

  • Animal-free (AOF) and carrier protein-free

  • Manufactured in Cambridge, UK

  • Lyophilized from HEPES/NaCl

  • Resuspend in 10 mM HCl, prepare single-use aliquots, add carrier protein if desired, and store frozen at -20oC or -80oC

Featured applications

  • Neural induction of human induced pluripotent stem cells

  • Differentiation of iPSC-derived neurons

  • Neural stem cell culture

  • Induction of axonal growth

  • Differentiation of iPSC-derived Schwann cells and astrocytes

  • Culture of retinal cells

Ciliary Neurotrophic Factor, CNTF, HCNTF, Ciliary Neuronotrophic Factor

  1. Murakami, M., Kamimura, D. & Hirano, T. New IL-6 (gp130) family cytokine members, CLC/NNT1/BSF3 and IL-27. Growth Factors 22, 75–77 (2004).
  2. Richardson, P. M. Ciliary neurotrophic factor: a review. Pharmacol Ther 63, 187–198 (1994).
  3. Pasquin, S., Sharma, M. & Gauchat, J. F. Ciliary neurotrophic factor (CNTF): New facets of an old molecule for treating neurodegenerative and metabolic syndrome pathologies. Cytokine Growth Factor Rev 26, 507–515 (2015).
  4. Guo, H. et al. The Roles of Ciliary Neurotrophic Factor – from Neuronutrition to Energy Metabolism. Protein Pept Lett 29, 815–828 (2022).
  5. Arakawa, Y., Sendtner, M. & Thoenen, H. Survival effect of ciliary neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: comparison with other neurotrophic factors and cytokines. J Neurosci 10, 3507–3515 (1990).
  6. Shimazaki, T., Shingo, T. & Weiss, S. The Ciliary Neurotrophic Factor/Leukemia Inhibitory Factor/gp130 Receptor Complex Operates in the Maintenance of Mammalian Forebrain Neural Stem Cells. The Journal of Neuroscience 21, 7642 (2001).
  7. Si, Z. P. et al. CNTF and Nrf2 Are Coordinately Involved in Regulating Self-Renewal and Differentiation of Neural Stem Cell during Embryonic Development. iScience 19, 303 (2019).
  8. Sendtner, M., Carrol, P., Holtmann, B., Hughes, R. A. & Thoenen, H. Ciliary neurotrophic factor. J Neurobiol 25, 1436–1453 (1994).
  9. Wen, R., Tao, W., Li, Y. & Sieving, P. A. CNTF AND RETINA. Prog Retin Eye Res 31, 136 (2012).
  10. Ding, J. et al. Role of Ciliary Neurotrophic Factor in the Proliferation and Differentiation of Neural Stem Cells. Journal of Alzheimer’s Disease 37, 587–592 (2013).
  11. Zahir, T., Klassen, H. & Young, M. J. Effects of Ciliary Neurotrophic Factor on Differentiation of Late Retinal Progenitor Cells. Stem Cells 23, 424–432 (2005).
  12. Müller, A., Hauk, T. G. & Fischer, D. Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation. Brain 130, 3308–3320 (2007).
  13. Leibinger, M., Andreadaki, A., Diekmann, H. & Fischer, D. Neuronal STAT3 activation is essential for CNTF- and inflammatory stimulation-induced CNS axon regeneration. Cell Death & Disease 2013 4:9 4, e805–e805 (2013).
  14. Leibinger, M. et al. Neuroprotective and Axon Growth-Promoting Effects following Inflammatory Stimulation on Mature Retinal Ganglion Cells in Mice Depend on Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor. Journal of Neuroscience 29, 14334–14341 (2009).
  15. Sleeman, M. W., Anderson, K. D., Lambert, P. D., Yancopoulos, G. D. & Wiegand, S. J. The ciliary neurotrophic factor and its receptor, CNTFR alpha. Pharm Acta Helv 74, 265–272 (2000).
  16. Ip, N. Y. et al. CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130. Cell 69, 1121–1132 (1992).
  17. Kruttgen, A. et al. Human ciliary neurotrophic factor: a structure-function analysis. Biochemical Journal 309, 215 (1995).
  18. Mashanov, V. et al. Synergistic effect of CNTF and GDNF on directed neurite growth in chick embryo dorsal root ganglia. PLoS One 15, (2020).
  19. Emdad, L., D’Souza, S. L., Kothari, H. P., Qadeer, Z. A. & Germano, I. M. Efficient Differentiation of Human Embryonic and Induced Pluripotent Stem Cells into Functional Astrocytes. https://home.liebertpub.com/scd 21, 404–410 (2011).
  20. Hollmann, E. K. et al. Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells. Fluids Barriers CNS 14, 1–13 (2017).
  21. Ribecco-Lutkiewicz, M. et al. A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis. Scientific Reports 2018 8:1 8, 1–17 (2018).
  22. Pan, J. et al. Acellular nerve grafts supplemented with induced pluripotent stem cell-derived exosomes promote peripheral nerve reconstruction and motor function recovery. Bioact Mater 15, 272–287 (2022).
  23. Lin, H. W., Jain, M., Li, H. & Levison, S. W. Ciliary neurotrophic factor (CNTF) plus soluble CNTF receptor α increases cyclooxygenase-2 expression, PGE2 release and interferon-γ-induced CD40 in murine microglia. J Neuroinflammation 6, 1–13 (2009).
  24. Krencik, R. & Zhang, S. C. Directed Differentiation of Functional Astroglial Subtypes from Human Pluripotent Stem Cells. Nat Protoc 6, 1710 (2011).
  25. Perugini, J. et al. Biological Effects of Ciliary Neurotrophic Factor on hMADS Adipocytes. Front Endocrinol (Lausanne) 10, 493947 (2019).
  26. Ip, N. Y. et al. CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130. Cell 69, 1121–1132 (1992).
  27. Wang, A. et al. Derivation of Smooth Muscle Cells with Neural Crest Origin from Human Induced Pluripotent Stem Cells. Cells Tissues Organs 195, 5–14 (2011).
  28. Linker, R. A. et al. CNTF is a major protective factor in demyelinating CNS disease: A neurotrophic cytokine as modulator in neuroinflammation. Nature Medicine 2002 8:6 8, 620–624 (2002).
  29. Linker, R. A. et al. CNTF is a major protective factor in demyelinating CNS disease: A neurotrophic cytokine as modulator in neuroinflammation. Nature Medicine 2002 8:6 8, 620–624 (2002).
  30. Leibinger, M. et al. Neuroprotective and Axon Growth-Promoting Effects following Inflammatory Stimulation on Mature Retinal Ganglion Cells in Mice Depend on Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor. Journal of Neuroscience 29, 14334–14341 (2009).
  31. Masu, Y. et al. Disruption of the CNTF gene results in motor neuron degeneration. Nature 1993 365:6441 365, 27–32 (1993).
  32. Tossetta, G. et al. Ciliary Neurotrophic Factor Modulates Multiple Downstream Signaling Pathways in Prostate Cancer Inhibiting Cell Invasiveness. Cancers (Basel) 14, 5917 (2022).

Bioactivity

Bioactivity graph showing the EC50 of 0.41 ng/ml (18 pM) for Qkine recombinant CNTF

CNTF activity is determined using the CNTF-responsive firefly luciferase reporter assay. HEK293T cells are treated in triplicate with a serial dilution of CNTF overnight. Firefly luciferase activity is measured and normalised to the control Renilla luciferase activity.

EC50 = 0.41 ng/ml (18 pM).

Data from Qk063 lot #204526.

Purity

Recombinant CNTF migrates as a major band at approximately 22.8 kDa (monomer) in non-reducing (NR) conditions. The dimeric form is the minor band at the higher molecular weight (45.6 kDa). Upon reduction (R), only the 22.8 kDa band is visible. No contaminating protein bands are present.

The purified recombinant protein (3 µg) was resolved using 15% w/v SDS-PAGE in reduced (+β-mercaptoethanol, R) and non-reduced (NR) conditions and stained with Coomassie Brilliant Blue R250. Data from Qk063 batch #204526.

Further quality assays

  • Mass spectrometry, single species with the expected mass
  • Endotoxin: <0.005 EU/μg protein (below the level of detection)
  • Recovery from stock vial: >95%

We are a company founded and run by scientists to provide a service and support innovation in stem cell biology and regenerative medicine.  All our products are exceptionally high purity, with complete characterisation and bioactivity analysis on every lot.

Protein background

Ciliary Neurotrophic Factor (CNTF) is a member of the IL-6 family of cytokines which also includes IL-6, IL-11, leukemia inhibitory factor (LIF), and Neuropoieitin [1-4]. It was initially isolated from chicken embryo ciliary neurons [4,5]. CNTF plays a crucial role in developing and maintaining the nervous and optic nervous systems [3,4,6,7]. It promotes the neurogenesis, differentiation, and survival of various neurons such as neural stem cells, sensory neurons, sympathetic neurons, and motor neurons, as well as oligodendrocytes, microglial cells, liver cells, skeletal muscle cells, and retinal cells [3,4,8–11]. Similarly, to LIF, CNTF is considered an inflammatory stimulation mediator with regenerative and axon growth-stimulating effects [12–14].

CNTF is a homodimer with a molecular weight of approximately 22.8 kDa and forms a four-helix bundle structure through hydrophobic interactions between the helices [2]. Each dimer is composed of 200 amino acids[2]. It is expressed by Schwann cells and astrocytes and binds to specific receptor complexes[2,3,15]. The complexes are composed of either the CNTF R alpha receptor subunit or the IL-6 R alpha receptor subunit with the recruitment of the gp130/LIFRβ receptor subunits[16]. This leads to the activation of various signaling pathways which promote cell survival, differentiation, and regeneration: JAK/STAT3 and PI3K/AKT/mTOR pathways[3,4,13,15].

As CNTF regulates neural development, it is used in vitro to initiate neural induction of human induced pluripotent stem cells (hiPSC) and differentiation of neural progenitor and neural stem cells along with IL-6, LIF, and oncostatin M[6,7,10].  CNTF promotes the self-renewal of neural stem cells with or without EGF, the survival of sensory and motor neurons, and neurite outgrowth[2,17,18]. CNTF also favours a cholinergic phenotype in sympathetic neurons[2,19–22].  Furthermore, CNTF can be used to promote the survival, differentiation, and maturation of glial cells such as astrocytes and Schwann cells. For example, Lin et al. showed that CNTF can activate microglia and dendritic-like microglia similarly to IL-6[23]. Also, Krencik and Zhang describe how to differentiate hiPSC-derived neuroepithelial cells into astrocytes using E8 media supplemented with EGF, FGF-2, and CNTF[24]. Finally, Pan et al. used CNTF with neuregulin 1β and dibutyryl cyclic AMP to obtain Schwann cells[22]. In addition, CNTF has also been reported in the culture of retinal cells and adipocytes[11,25–27].

Due to its protective and regenerative role in the nervous system, research on CNTF has been focused on neurodegenerative diseases (amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease) to slow down the disease progression, as well as retinal diseases (retinitis pigmentosa, and macular degeneration)[4,28–31]. CNTF has also been investigated to treat peripheral nerve injuries and chronic inflammatory diseases (multiple sclerosis) to enhance nerve regeneration and promote neurite growth[29]. The therapeutic potential of CNTF is also extended to treat metabolic diseases to regulate the balance of energy metabolism and induce body weight loss[3,4]. Finally, CNTF has been shown to be involved as a negative modulator of invasion processes of prostate cancer in an in vitro model[32].

Additional resources

Technote: CNTF

Our products are for research use only and not for diagnostic or therapeutic use.  Products are not for resale.

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