A method for the generation of large numbers of dendritic cells from CD34+ hematopoietic stem cells from cord blood

Nicole Bedke, Emily J. Swindle, Camelia Molnar, Patrick G. Holt, Deborah H. Strickland, Graham C. Roberts, Ruth Morris, Stephen T. Holgate, Donna E. Davies, Cornelia Blume

Research output: Contribution to journalArticle

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-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-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.

Original languageEnglish
Article number112703
JournalJournal of Immunological Methods
DOIs
Publication statusE-pub ahead of print - 9 Nov 2019

Fingerprint

Hematopoietic Stem Cells
Fetal Blood
Dendritic Cells
Interleukin-6
Interleukin-3
Adaptive Immunity
Virus Diseases
Granulocyte-Macrophage Colony-Stimulating Factor
Interleukin-1
Bacterial Infections
Innate Immunity
Interleukin-4
Interleukin-10
Blood Cells

Cite this

Bedke, Nicole ; Swindle, Emily J. ; Molnar, Camelia ; Holt, Patrick G. ; Strickland, Deborah H. ; Roberts, Graham C. ; Morris, Ruth ; Holgate, Stephen T. ; Davies, Donna E. ; Blume, Cornelia. / A method for the generation of large numbers of dendritic cells from CD34+ hematopoietic stem cells from cord blood. In: Journal of Immunological Methods. 2019.
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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-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-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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A method for the generation of large numbers of dendritic cells from CD34+ hematopoietic stem cells from cord blood. / Bedke, Nicole; Swindle, Emily J.; Molnar, Camelia; Holt, Patrick G.; Strickland, Deborah H.; Roberts, Graham C.; Morris, Ruth; Holgate, Stephen T.; Davies, Donna E.; Blume, Cornelia.

In: Journal of Immunological Methods, 09.11.2019.

Research output: Contribution to journalArticle

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T1 - A method for the generation of large numbers of dendritic cells from CD34+ hematopoietic stem cells from cord blood

AU - Bedke, Nicole

AU - Swindle, Emily J.

AU - Molnar, Camelia

AU - Holt, Patrick G.

AU - Strickland, Deborah H.

AU - Roberts, Graham C.

AU - Morris, Ruth

AU - Holgate, Stephen T.

AU - Davies, Donna E.

AU - Blume, Cornelia

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Y1 - 2019/11/9

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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-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.

KW - CD34+ hematopoietic stem cells

KW - Cord blood

KW - Lipopolysaccharide

KW - Maturation

KW - Neonatal dendritic cells

KW - Poly(I:C)

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