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.

中文翻译：

---

一种从脐带血的 CD34+ 造血干细胞中产生大量树突状细胞的方法。

树突状细胞 (DC) 在调节先天性和适应性免疫反应中发挥着核心作用。人们普遍认为，它们的调节功能在生命过程中会发生变化。为了研究早期 DC 的功能，重要的是表征新生儿 DC 的功能。然而，由于伦理原因或脐带血样本相对较少，新生儿 DC 的可用性受到限制，因此难以进行大规模实验。我们的目标是建立一个稳健的方案，用于从脐血来源的 CD34+ 造血干细胞生成新生儿 DC。对于 DC 前体细胞的扩增，我们使用了含有 Flt-3 L、SCF、TPO、IL-3 和 IL-6 的细胞因子混合物。在扩增培养的前 2 周内 IL-3 和 IL-6 的存在对于表达 CD14 的 DC 前体细胞的增殖至关重要。培养 4 周后，选择 CD14+ 前体细胞并在 GM-CSF 和 IL-4 存在下产生功能性 DC。然后用 Poly(I:C) 和 LPS 刺激新生儿 DC，以分别模拟病毒或细菌感染。与 LPS 相比，Poly(I:C) 诱导了成熟标志物 CD80、CD86 和 CD40 的更高表达。与使用脐带血 DC 的文献数据一致，与 LPS 成熟的新生儿 DC 相比，我们的 Poly(I:C) 成熟的新生儿 DC 细胞显示出更高的 IL-12p70 释放。此外，与源自脐血 CD34+ 的 LPS 成熟新生儿 DC 相比，我们证明了 IFN-γ、TNF-α、IL-1β 和 IL-6 的更高释放，但在 Poly(I:C) 中 IL-10 的释放更低造血干细胞。总之，我们为生成大量功能性新生儿 DCs 建立了一个强大的协议。