PMID- 31711888 OWN - NLM STAT- MEDLINE DCOM- 20200929 LR - 20220302 IS - 1872-7905 (Electronic) IS - 0022-1759 (Print) IS - 0022-1759 (Linking) VI - 477 DP - 2020 Feb TI - A method for the generation of large numbers of dendritic cells from CD34+ hematopoietic stem cells from cord blood. PG - 112703 LID - S0022-1759(19)30041-9 [pii] LID - 10.1016/j.jim.2019.112703 [doi] LID - 112703 AB - 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-3 L, 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-gamma, TNF-alpha, IL-1beta 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. CI - Copyright (c) 2019 The Authors. Published by Elsevier B.V. All rights reserved. FAU - Bedke, Nicole AU - Bedke N AD - Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK. FAU - Swindle, Emily J AU - Swindle EJ AD - Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK. FAU - Molnar, Camelia AU - Molnar C AD - Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK. FAU - Holt, Patrick G AU - Holt PG AD - Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Perth, Australia. FAU - Strickland, Deborah H AU - Strickland DH AD - Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Perth, Australia. FAU - Roberts, Graham C AU - Roberts GC AD - Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK. FAU - Morris, Ruth AU - Morris R AD - Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK. FAU - Holgate, Stephen T AU - Holgate ST AD - Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK. FAU - Davies, Donna E AU - Davies DE AD - Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK. FAU - Blume, Cornelia AU - Blume C AD - Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK. Electronic address: C.Blume@soton.ac.uk. LA - eng GR - G0800766/MRC_/Medical Research Council/United Kingdom GR - G0900453/MRC_/Medical Research Council/United Kingdom PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20191109 PL - Netherlands TA - J Immunol Methods JT - Journal of immunological methods JID - 1305440 RN - 0 (Adjuvants, Immunologic) RN - 0 (Antigens, CD34) RN - 0 (Culture Media) RN - 0 (Cytokines) RN - 0 (Lipopolysaccharides) RN - 83869-56-1 (Granulocyte-Macrophage Colony-Stimulating Factor) RN - O84C90HH2L (Poly I-C) SB - IM MH - Adjuvants, Immunologic/pharmacology MH - Antigens, CD34/metabolism MH - Bacterial Infections/immunology MH - Cell Count MH - Cell Differentiation/drug effects MH - Cells, Cultured MH - Cesarean Section MH - Culture Media/metabolism MH - Cytokines/metabolism MH - Dendritic Cells MH - Female MH - Fetal Blood/*cytology MH - Granulocyte-Macrophage Colony-Stimulating Factor/metabolism MH - Hematopoietic Stem Cells/drug effects/metabolism/*physiology MH - Humans MH - Infant, Newborn MH - Lipopolysaccharides/immunology MH - Poly I-C/immunology MH - Primary Cell Culture/*methods MH - Virus Diseases/immunology PMC - PMC6983936 OTO - NOTNLM OT - CD34+ hematopoietic stem cells OT - Cord blood OT - Lipopolysaccharide OT - Maturation OT - Neonatal dendritic cells OT - Poly(I:C) EDAT- 2019/11/13 06:00 MHDA- 2020/09/30 06:00 PMCR- 2020/02/01 CRDT- 2019/11/13 06:00 PHST- 2019/03/19 00:00 [received] PHST- 2019/11/06 00:00 [revised] PHST- 2019/11/07 00:00 [accepted] PHST- 2019/11/13 06:00 [pubmed] PHST- 2020/09/30 06:00 [medline] PHST- 2019/11/13 06:00 [entrez] PHST- 2020/02/01 00:00 [pmc-release] AID - S0022-1759(19)30041-9 [pii] AID - 112703 [pii] AID - 10.1016/j.jim.2019.112703 [doi] PST - ppublish SO - J Immunol Methods. 2020 Feb;477:112703. doi: 10.1016/j.jim.2019.112703. Epub 2019 Nov 9.