A method for the generation of large numbers of dendritic cells from CD34+ hematopoietic stem cells from cord blood
A method for the generation of large numbers of dendritic cells from CD34+ hematopoietic stem cells from cord blood
Dendritic cells (DCs) play a central role in regulating innate and adaptive immune responses. It is well accepted that their regulatory functions change over the life course. In order to study DCs function during early life it is important to characterize the function of neonatal DCs. However, the availability of neonatal DCs is limited due to ethical reasons or relative small samples of cord blood making it difficult to perform large-scale experiments. Our aim was to establish a robust protocol for the generation of neonatal DCs from cord blood derived CD34+ hematopoietic stem cells. For the expansion of DC precursor cells we used a cytokine cocktail containing Flt-3L, SCF, TPO, IL-3 and IL-6. The presence of IL-3 and IL-6 in the first 2 weeks of expansion culture was essential for the proliferation of DC precursor cells expressing CD14. After 4 weeks in culture, CD14+ precursor cells were selected and functional DCs were generated in the presence of GM-CSF and IL-4. Neonatal DCs were then stimulated with Poly(I:C) and LPS to mimic viral or bacterial infections, respectively. Poly(I:C) induced a higher expression of the maturation markers CD80, CD86 and CD40 compared to LPS. In line with literature data using cord blood DCs, our Poly(I:C) matured neonatal DCs cells showed a higher release of IL-12p70 compared to LPS matured neonatal DCs. Additionally, we demonstrated a higher release of IFN-γ, TNF-α, IL-1β and IL-6, but lower release of IL-10 in Poly(I:C) matured compared to LPS matured neonatal DCs derived from cord blood CD34+ hematopoietic stem cells. In summary, we established a robust protocol for the generation of large numbers of functional neonatal DCs. In line with previous studies, we showed that neonatal DCs generated form CD34+ cord blood progenitors have a higher inflammatory potential when exposed to viral than bacterial related stimuli.
CD34+ hematopoietic stem cells, Poly(I:C), cord blood, lipopolysaccharide, maturation, neonatal dendritic cells
1-8
Bedke, Nicole J
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Swindle, Emily
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Molnar, Camelia
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Holt, Patrick G
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Strickland, Deborah H.
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Roberts, Graham
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Morris, Ruth C.G.
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Holgate, Stephen
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Davies, Donna
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Blume, Cornelia
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February 2020
Bedke, Nicole J
981dbd61-1912-4231-b6d5-42520c38178d
Swindle, Emily
fe393c7a-a513-4de4-b02e-27369bd7e84f
Molnar, Camelia
39b09914-aaf1-4997-b032-4dee91060d40
Holt, Patrick G
e1b8fc2d-2c79-4674-a6bf-f4b047e6164f
Strickland, Deborah H.
69b2bdae-32a6-4b9a-8cfd-d9cfb93dbd91
Roberts, Graham
ea00db4e-84e7-4b39-8273-9b71dbd7e2f3
Morris, Ruth C.G.
765a2381-c682-4221-a3c1-a7531de7fdff
Holgate, Stephen
2e7c17a9-6796-436e-8772-1fe6d2ac5edc
Davies, Donna
7de8fdc7-3640-4e3a-aa91-d0e03f990c38
Blume, Cornelia
aa391c64-8718-4238-906b-d6bb1551a07b
Bedke, Nicole J, Swindle, Emily, Molnar, Camelia, Holt, Patrick G, Strickland, Deborah H., Roberts, Graham, Morris, Ruth C.G., Holgate, Stephen, Davies, Donna and Blume, Cornelia
(2020)
A method for the generation of large numbers of dendritic cells from CD34+ hematopoietic stem cells from cord blood.
Journal of Immunological Methods, 477, , [112703].
(doi:10.1016/j.jim.2019.112703).
Abstract
Dendritic cells (DCs) play a central role in regulating innate and adaptive immune responses. It is well accepted that their regulatory functions change over the life course. In order to study DCs function during early life it is important to characterize the function of neonatal DCs. However, the availability of neonatal DCs is limited due to ethical reasons or relative small samples of cord blood making it difficult to perform large-scale experiments. Our aim was to establish a robust protocol for the generation of neonatal DCs from cord blood derived CD34+ hematopoietic stem cells. For the expansion of DC precursor cells we used a cytokine cocktail containing Flt-3L, SCF, TPO, IL-3 and IL-6. The presence of IL-3 and IL-6 in the first 2 weeks of expansion culture was essential for the proliferation of DC precursor cells expressing CD14. After 4 weeks in culture, CD14+ precursor cells were selected and functional DCs were generated in the presence of GM-CSF and IL-4. Neonatal DCs were then stimulated with Poly(I:C) and LPS to mimic viral or bacterial infections, respectively. Poly(I:C) induced a higher expression of the maturation markers CD80, CD86 and CD40 compared to LPS. In line with literature data using cord blood DCs, our Poly(I:C) matured neonatal DCs cells showed a higher release of IL-12p70 compared to LPS matured neonatal DCs. Additionally, we demonstrated a higher release of IFN-γ, TNF-α, IL-1β and IL-6, but lower release of IL-10 in Poly(I:C) matured compared to LPS matured neonatal DCs derived from cord blood CD34+ hematopoietic stem cells. In summary, we established a robust protocol for the generation of large numbers of functional neonatal DCs. In line with previous studies, we showed that neonatal DCs generated form CD34+ cord blood progenitors have a higher inflammatory potential when exposed to viral than bacterial related stimuli.
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accepted manuscript
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More information
Accepted/In Press date: 7 November 2019
e-pub ahead of print date: 9 November 2019
Published date: February 2020
Additional Information:
Funding Information:
This work was funded by the Medical Research Council UK ( G0900453 ). Appendix A
Publisher Copyright:
© 2019 The Authors
Keywords:
CD34+ hematopoietic stem cells, Poly(I:C), cord blood, lipopolysaccharide, maturation, neonatal dendritic cells
Identifiers
Local EPrints ID: 435812
URI: http://eprints.soton.ac.uk/id/eprint/435812
ISSN: 0022-1759
PURE UUID: 301c0461-415b-42d3-8f2c-fe7f957a5c2a
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Date deposited: 21 Nov 2019 17:30
Last modified: 17 Mar 2024 03:19
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Contributors
Author:
Nicole J Bedke
Author:
Camelia Molnar
Author:
Patrick G Holt
Author:
Deborah H. Strickland
Author:
Ruth C.G. Morris
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