Optimization of seeding density of OP9 cells to improve hematopoietic differentiation efficiency.

Background: 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.

Methods: 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.

Results: 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 × 10⁴ cells/cm² 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 × 10⁴ cells/cm² 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.

Conclusion: The peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 10⁶ cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency.