Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, USA.
Cytotherapy. 2010 Oct;12(6):767-82. doi: 10.3109/14653241003786148.
Megakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization and platelet release. pH and pO2 increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks.
CD34+ HSPCs were cultured at pH 7.2 and 5% O2 with stem cell factor (SCF), thrombopoietin (Tpo) and all combinations of Interleukin (IL)-3, IL-6, IL-11 and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O2 to generate mature Mks, and treated with nicotinamide (NIC) to enhance polyploidization.
Using Tpo + SCF + IL-3 + IL-11, we obtained 3.5 CD34+ CD41+ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO2. Although fewer progenitors were present, shifting to 20% O2 /pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. NIC more than doubled the percentage of high-ploidy Mks to 40%.
We obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed NIC addition will greatly increase high-ploidy Mk production.
巨核细胞生成包括造血干细胞和祖细胞（HSPC）向巨核细胞（Mk）谱系的定向分化、Mk 祖细胞和成熟 Mk 的扩增、多倍体化和血小板释放。pH 值和氧分压从骨内膜到窦状隙逐渐增加，不同的细胞因子对于分化的不同阶段很重要。我们假设，在骨髓中模拟 Mk 分化过程中的变化条件，将有助于祖细胞的扩增，从而产生许多高倍体的 Mk。
CD34+ HSPC 在 pH 值为 7.2 和 5% O2 条件下，与干细胞因子（SCF）、血小板生成素（Tpo）以及白细胞介素（IL）-3、IL-6、IL-11 和 Flt-3 配体的各种组合联合培养，以促进 Mk 祖细胞的扩增。用选定的细胞因子培养的细胞被转移到 pH 值为 7.4 和 20% O2 以生成成熟的 Mk，并使用烟酰胺（NIC）增强多倍体化。
使用 Tpo + SCF + IL-3 + IL-11，我们从每个输入的 HSPC 中获得了 3.5 个 CD34+ CD41+ Mk 祖细胞，同时纯度从 1%提高到 17%。含有 IL-3 的细胞因子鸡尾酒产生了更多的祖细胞和成熟的 Mk，但纯度较低。在较高的 pH 值和氧分压下，Mk 的产生量更大。虽然祖细胞数量较少，但在第 5 天（而非第 7 天或第 9 天）将氧分压和 pH 值分别调整为 20%和 7.4，可产生最多的成熟 Mk，每个输入的 HSPC 可产生 14 个 Mk。NIC 将高倍体 Mk 的比例增加了一倍多，达到 40%。
我们获得了广泛的 Mk 祖细胞扩增，同时确保祖细胞能够产生高倍体的 Mk。我们预计，随后对成熟 Mk 生成的细胞因子进行优化，并延迟 NIC 的添加，将大大增加高倍体 Mk 的生成。
