Abstract: Objective To investigate the regulatory role of protein kinase A (PKA) in the differentiation of human induced pluripotent stem cells (iPSCs) into megakaryocytes (MKs). Methods A PKA overexpression plasmid was constructed and introduced into iPSCs derived from human umbilical cord blood via a lentiviral transduction system to establish a PKA overexpression cell line. A PKA gene knockout iPSC line was generated using CRISPR/Cas9 gene-editing technology. The expression of PKA in both cell lines was verified at the protein level. In a serum-free, feeder-free "Spin-EB" culture system, three groups were established: control group (untreated group), PKA overexpression group (OV group), and PKA knockout group (KO group). On day 21 of culture, cells from each group were collected, and the expression levels of megakaryocytic lineage markers (CD41⁺) and mature megakaryocytic markers (CD41⁺CD42a⁺) were detected by flow cytometry. Results No significant difference was observed in CD41⁺ cell proportion or count between PKA OV and control groups (P>0.05); PKA OV significantly reduced the proportion and count of CD41⁺CD42a⁺ mature MKs (P<0.01; P<0.05); PKA KO markedly increased both CD41⁺ and CD41⁺CD42a⁺ cell proportions and counts (P<0.001; P<0.001; P<0.01; P<0.01). Conclusion This study successfully established PKA gene-modified iPSC lines and demonstrated the regulatory role of PKA in the differentiation of iPSCs into MKs, providing a new target for optimizing the generation of platelets from human iPSCs. PKA overexpression inhibited the differentiation of iPSCs into MKs, whereas PKA knockout significantly promoted this differentiation.

Key words: Human induced pluripotent stem cells, In vitro differentiation, Megakaryocytes, Protein kinase A