The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan Province, China.
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Hainan, China.
BMC Mol Cell Biol. 2024 Mar 25;25(1):10. doi: 10.1186/s12860-024-00503-x.
OP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However, the whole co-culture procedure usually needs 14-18 days, including preparing OP9 cells at least 4 days. Therefore, the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency.
In the experimental group, we set six different densities of OP9 cells and just cultured them for 24 h before co-culture, and in the control group, OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them.
OP9 cells were randomly assigned into two groups. In the experimental group, six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast, in the control group, OP9 cells were cultured for 4 days at a total number of 3.1 × 10 cells/cm in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining, and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 10 cells/cm in a 6-well plate which was cultured just for 1 day, followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group.
The peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 10 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency.
OP9 鼠基质细胞系已被广泛用于诱导人胚胎干细胞（hESC）分化为造血干/祖细胞（HSPC）。然而，整个共培养过程通常需要 14-18 天，包括至少 4 天的 OP9 细胞准备。因此，这种低效的分化系统不受欢迎。我们旨在优化培养条件以提高分化效率。
在实验组中，我们设置了 OP9 细胞的六个不同密度，并在共培养前仅培养 24 小时，而在对照组中，OP9 细胞在共培养前培养 4 天以达到过度生长状态。然后，我们比较了它们之间的造血分化效率。
OP9 细胞被随机分为两组。在实验组中，六个不同接种数目的 OP9 细胞在共培养前培养 1 天。相比之下，在对照组中，OP9 细胞在 6 孔板中以总数为 3.1×10 个细胞/cm2培养 4 天以达到过度生长状态，然后进行共培养。通过 CD34 免疫染色评估造血分化，并比较这两组之间的差异。我们无法影响在 6 孔板中以总数为 10.4×10 个细胞/cm2培养 1 天的 OP9 细胞的分化效率，随后与 hESC 共培养。与对照组相比，它可以提前 5 天达到相同的分化效率。
与对照组相比，CD34+细胞的峰值出现早了 2 天。OP9 细胞在 6 孔板中的总数为 1.0×10 个细胞对于获得高分化效率是合适的。
