Bulk RNA-seq study 8- RNA-seq of male KOLF2.2J hiPSC-derived trophoblast cell lines homozygous null for nine different transcription factors.

This study aims to phenotype iPSC-derived trophoblast lines that contain homozygous null alleles for transcription factors expressed within the extra-embryonic lineage, with a focus on differentiation toward either primitive syncytium or extra-embryonic mesenchymal cells, depending on the TF being analyzed. Null alleles were generated by insertion of a premature termination codon with frameshift (PTC+1). RNA-seq data was generated after 6 days of initiating differentiation from iPSCs. The study included the homozygous null alleles for the following transcription factors: DLX3, MSX2, VGLL1, MAFB, NR2F2, ELF5, ASCL2, HIF1A, and TP63. Notably, the hypoxia-inducible factor HIF1A as well as ELF5, ASCL2, and TP63 were evaluated under both 20% oxygen and 3% (~ level present at peri-implantation) oxygen concentrations. This analysis provided insight into the impact of loss of function for these transcription factors in trophoblast lineage differentiation. The study involved the generation of RNA-seq on 53 samples. Eight of the transcription factors were analyzed in the primitive syncytium differentiation scheme and one in the extra-embryonic mesenchymal cell scheme. Three biological replicates were run for each gene. Additionally, three biological replicates were run of wild-type KOLF2.2J cells (the parental male cell line in which the KOs were generated) under the same differentiation conditions and under both 20% and, in some cases, 3% O₂ concentrations. This comprehensive design allowed for a robust analysis on the effects of transcription factor manipulation on cell fate decisions within the extra-embryonic lineage.